HU205627B - Process for producing polypeptides by yeasts containing area for controlling gen expression - Google Patents

Abstract

Novel DNA sequences which are responsive to the presence of methanol, catabolite non-repressing carbon sources and carbon source starvation are provided. In addition, novel constructs including these DNA sequences, as well as transformed organisms therewith are provided. Processes for producing the DNA sequences and constructs of the invention are detailed. The production of polypeptide product under the control of the regulatory regions of the invention is demonstrated.

Description

The present invention relates to the field of recombinant DNA biotechnology. In one aspect, the invention relates to DNA fragments that control the transcription of DNA into messenger RNA and the initiation and termination of translation (translation) of messenger RNA into protein. In another aspect, the invention relates to expression vectors that include the DNA fragments described above. In yet another aspect, the invention relates to novel microorganisms transformed with the expression vectors described above. In another aspect, the invention relates to the production of polypeptides.

As recombinant DNA technology has evolved in recent years, microorganisms have been able to regulate the production of a huge variety of useful polypeptides. More eukaryotic polypeptides, e.g. human growth hormone, leukocyte interferons, human insulin and human proinsulin are already produced by various microorganisms. The continued use of techniques already in the art is expected in the future to allow the production of additional useful polypeptides by microorganisms.

Basic techniques of recombinant DNA technology are known to those of skill in the art. For the practical application of recombinant DNA technology in microorganism hosts, it is preferred that the following elements be present (without limitation):

(1) a gene encoding one or more desired polypeptides provided with appropriate regulatory sequences necessary for expression in the host microorganism.

(2) a vector, usually a plasmid, into which the gene can be inserted. The vector serves to ensure the transfer of the gene to the cell and the retention of DNA sequences in the cell, as well as a high level of expression of the aforementioned genes, and (3) an appropriate host microorganism into which the vector carrying the desired gene can be transformed, the host microorganism also has a cell apparatus that allows expression of the information encoded by the inserted gene.

One of the basic elements used in recombinant DNA technology is the plasmid, which is an extrachromosomal double-stranded DNA found in certain microorganisms. Where plasmids are naturally occurring in microorganisms, they often occur at multiple copy numbers per cell. In addition to the naturally occurring plasmids, a variety of human-made plasmids or hybrid vectors have been produced. The information encoded in plasmid DNA also includes the information necessary for the plasmid to be reproduced in daughter cells, i.e., an autonomously replicating sequence or origin of replication. One or more phenotypic selection traits must also be present in the information encoded in the plasmid DNA. The phenotypic selection characteristics allow the recognition and selection of clones of a host cell containing the plasmid of interest by growing the cells in selective media.

The utility of plasmids is based on the fact that they can be specifically cleaved by one or another restriction endonuclease or restriction enzyme, each of which recognizes a specific, unique site on the plasmid DNA. The homologous genes, heterologous genes, i.e. genes or gene fragments from non-host organisms, can then be inserted into the plasmid (-) by linking the ends of the cleaved plasmid and the desired genetic material (at the cleavage site or at the end of the cleavage site). The resulting recombinant DNA material may be called a hybrid vector.

DNA recombination is performed outside the host microorganism. The resulting hybrid vector is introduced into the host microorganism by a process known as transformation. By growing the transformed microorganism, a large amount of hybrid vectors can be obtained. If the gene is inserted appropriately for the transcriptional and translational control regions of the encoded DNA message, the resulting hybrid vector can be used to direct the production of the polypeptide sequence encoded by the inserted gene. Production of the polypeptide in this manner is termed gene expression.

Gene expression begins in the DNA region known as the promoter region. During the transcriptional phase of expression, DNA is twisted, serving as a template (template) for the synthesis of messenger RNA. The RNA polymerase binds to the promoter region and runs along the intact DNA from its 3 'end to its 5' end, transcribing the information contained in the coding strand into the messenger RNA (mRNA) from the 5 'end to the 3' end of the mRNA. The messenger RNA then binds to ribosomes, where the mRNA is translated into a polypeptide chain. Each amino acid is encoded by a nucleotide triplet or codon within a structure that can be termed a structural gene, a portion of the gene that encodes the amino acid sequence of the expressed product. Since the production of each amino acid is encoded by three nucleotides, there are three different ways to "read" the nucleotide sequence. The reading frame that encodes the desired polypeptide product is called the appropriate reading frame.

After binding to the promoter, the RNA polymerase first transcribes a 5 'leader region of the mRNA, then a translational initiation or start codon, followed by the nucleotide codons within the structural gene itself. In order to obtain the desired gene product, it is necessary that the start or start codon correctly (-) initiate translation of the messenger RNA by the ribosomes (in the appropriate reading frame). Finally, the stop codons are transcribed at the end of the structural gene, causing any additional mRNA sequences not to be transcribed by the ribosomes, even if additional mRNA sequences were formed by interaction of the RNA polymerase with the DNA template. Thus, stop codons determine the end of translation, and thus the end of further incorporation of amino acids into the polypeptide product. The polypeptide product is a host cell

This can be achieved by lysing the lysine and appropriate purification from other microbial proteins or, in some circumstances, purifying the fermentation medium in which the host cell has grown and into which the polypeptide product is excreted.

In practice, recombinant DNA technology can result in microorganisms that are capable of expressing completely heterologous polypeptides, that is, polypeptides that are normally not present or produced in a given microorganism - a so-called direct expression. However, it is also possible to express or produce a fusion protein, that is, a heterologous polypeptide fused to a portion of the amino acid sequence of a homologous polypeptide, wherein the homologous polypeptide is a polypeptide found in a wild-type (untransformed) microorganism - this is called indirect expression. In the case of indirect expression, the initially obtained fusion protein product is sometimes inactive for its intended use until the fused homologous / heterologous polypeptide is cleaved in an extracellular environment. so e.g. cyanogen bromide cleavage of methionine residues results in (-) somatostatin, thymosin alfa-1 and human insulin A and B chain components (from fused homologous / heterologous polypeptides), whereas enzymatic cleavage of the specified radicals results in beta-endorphin.

So far, industrial efforts to produce various polypeptides using recombinant DNA technology have focused on Escherichia coli as a host. However, in some situations, E. coli proved to be unfit to host. so e.g. E. coli contains a number of toxic pyrogenic factors that must be removed from any polypeptide used as a pharmaceutical product. Of course, the purification efficiency will vary with each polypeptide. In addition, the proteolytic activity of E. coli greatly limits the yield of certain useful products. These and other considerations have led to increased interest in other types of hosts, particularly the use of eukaryotic organisms for the production of polypeptide products. The polypeptide products are found in eukaryotic systems, e.g. The tools needed for yeast production have significant advantages over prokaryotic systems such as yeast. Compared with E. coli in the production of polypeptides encoded by recombinant DNA. Yeasts have been used for large-scale fermentations for centuries, whereas, in comparison, large-scale fermentations of E. coliferations have been relatively recent. Yeasts usually grow at a higher cell density than bacteria and are easily adapted to continuous fermentation processes. Growth of yeasts such as Pichia pastoris at high cell densities, i.e., above 100 g / l, is described by Wagner in U.S. Patent No. 4,414,329 (filed by Phillips Petroleum Co.). A further advantage of yeast hosts is that several critical functions of the organism, e.g. oxidative phosphorylation is located within the organelles and thus is not exposed to the potential detrimental effects of the production of a foreign polypeptide by wild-type host cells. Yeasts, being eukaryotic organisms, may be capable of glycosylating expressed polypeptide products, where such glycosylation is important for the bioactivity of the polypeptide product. It is also possible that, as a eukaryotic organism, yeasts prefer the same codons as higher organisms, such as those derived from mammalian genes or e.g. tend to produce more efficiently expressed products derived from complementary DNA (cDNA) obtained by reverse transcription from mammalian mRNA.

The prevalence of poorly characterized yeast strains such as host / vector systems is hampered by lack of transformation conditions and lack of knowledge of appropriate vectors. In addition, auxotrophic mutants are often unavailable, precluding the direct selection of transformants by auxotrophic complementation. To fully fulfill the promise of recombinant DNA technology, new host / vector systems must be constructed that facilitate DNA manipulation and optimal expression of the inserted DNA sequences so that the desired polypeptide product can be produced under controlled conditions and in high yield.

In the development of the present invention, DNA sequences have been discovered that regulate DNA transcription into messenger RNA and translation of messenger RNA resulting in a polypeptide. According to the present invention, novel DNA sequences can be used to produce polypeptide products

(a) by yeast cells capable of growing on methanol as the sole carbon and energy source;

(b) by yeast cells capable of growing on glucose, ethanol, fructose and the like; and

(c) by yeast cells capable of growing on glycerol, galactose, acetate and the like.

The following is a brief description of the figures:

Figure 1 shows a comparison between the genome clone of p76 protein (pPG 6.10) (a) and cDNA clone (pPC 15.0) (b).

Figure 2 shows a comparison of the p72 protein genome clone (pPG 4.0) (a) and cDNA clone (pPC 8.3) (b) (alcohol oxidase) and (pPC 8.0) cDNA clone (e).

Figure 3 shows a comparison of the genome clone (pPG 4.8) (a) and cDNA clone (pPC 6.7) (b) of the p40 protein ·

Figure 4 shows a restriction map (b) and a map of structural sequences (a) of the regulatory region of pPG 6.0 according to the invention.

Figure 5 is a restriction map of the regulatory region of pPG 4.0 according to the invention.

Figure 6 shows a restriction map of the regulatory region of pPG clone 4.8 according to the invention.

Figure 7 is a restriction map of the DNA sequence obtained from the 3 'end of the p76 structural gene.

HU 205 627 Β

Figure 8 shows a restriction map of the DNA sequences obtained from the 3 'end of the p72 (alcohol oxidase) structural gene. (a: 0.2 kb StuI-PvuII fragment, b: 0, kb StuI-HindIII fragment; c: 3.2 kb SalI-EcoRI fragment).

Figure 9 is a restriction map of the DNA sequence obtained from the 3 'end of the p40 structural gene.

Figure 10 is a restriction map of the p76 structural gene and its 5 'regulatory region.

All. Figure 3B is a restriction map of the p40 protein structural gene and its regulatory region 5 '.

A12. Figure 3B is a cDNA restriction map of p76 protein.

Figure 13 is a cDNA restriction map of p72 protein (alcohol oxidase).

A14. Figure 3B is a cDNA restriction map of p40 protein.

A15. Fig. 2A shows a restriction map of the lacZ DNA construct of the two novel p76 regulatory regions of the invention (a: 0.85 kbp from HindIII-BamHI from the regulatory region of pPG 6.05 'and lacZ gene from E. coli; b: 1.3 kbp from the regulatory region of pPG 6.05' HindIII-EcoRI and the lacZ gene from E. coli).

Figure 16 is a novel lacZ DNA construct of the novel p72 (alcohol oxidase) regulatory region of the present invention.

A17. Figure 3B is a restriction map of plasmid pSAOH1.

A18. Figure 3B is a restriction map of plasmid pSAOH 5.

A19. Figure 3B is a restriction map of plasmid pSAOH 10.

Figure 20 is a restriction map of plasmid pTAFH.85.

Figure 21 is a restriction map of plasmid pT76H1.

Figure 22 is a restriction map of plasmid pT76H2.

Figure 22a is a restriction map of plasmid pT76H3.

Figure 22 b is a restriction map of plasmid p76H4.

Figure 23 is a restriction map of plasmid pYA2.

Figure 24 is a restriction map of plasmid pYA4.

Figure 25 is a restriction map of plasmid pYJ8.

Figure 26 is a restriction map of plasmid pYJ8 ACla.

Figure 27 is a restriction map of plasmid pYJ30.

Figure 28 is a restriction map of plasmid pTAFH1 showing how the plasmid was constructed.

Figure 29 is a restriction map of plasmid pTAO 12 showing how the plasmid was constructed.

Figure 30 is a restriction map of plasmid pTAO 13.

Figure 30 (a) is a restriction map of plasmid pT76U1.

Figure 31 is a restriction map of plasmid pTAO1 showing how the plasmid was constructed.

Figure 32 is a restriction map of plasmid pTAF.85 showing how the plasmid was constructed.

Figure 33 provides a restriction map of plasmid YEpl3.

Figure 34 is a restriction map of plasmid pBP11.

The restriction enzymes used in the method of the invention are abbreviated as follows:

A = Asuü

B = BamHI

B ₂ = BglII

B _c = Bell

C = Clal

H ₂ = HincII

H ₃ = Hindyl

K = KpnI

Nd, = Ndel

No = NruI ps = PstI p _Vl = PvuI Pv ₂ = PvuII Ri = Eco r ₅ = EcoRV R _s = RsaI S = Sáli s ₃ = Sau3AI Neither = Sac sm = Smal sp = SphI ss = Sst St = Stu T = Taq th = thai Xb - XbaI xH = XhoI xm - XmaI

In the figures, restriction sites which are used during manipulation of DNA fragments but disappear during ligation are indicated by brackets.

The present invention therefore relates to a process for the preparation of a novel DNA fragment comprising a regulatory domain sensitive to at least one of the following conditions: presence of methanol, carbon source when cells grow on certain substrates other than methanol, and non-catabolite repressing carbon sources other than methanol. The regulatory region of the DNA fragment of the invention is capable of regulating transcription of messenger RNA when positioned at the 5 'end of a DNA encoding the production of messenger RNA. Mutants of the DNA fragments described above are also within the scope of the present invention.

The DNA fragment produced by the method of the invention further comprises a regulatory region capable of controlling polyadenylation, termination of transcription, and termination of translation of messenger RNA when located at the 3 'end of the polypeptide coding region that expresses messenger RNA. encoding, whereby transcription and translation of messenger RNA is regulated by a regulatory region sensitive to at least one of the following conditions: presence of methanol, absence of carbon source when cells grow on certain substrates other than methanol, and presence of non-catabolite repressing carbon sources other than methanol. Mutants of the DNA fragments described above are also within the scope of the present invention.

In a particular embodiment of the method of the invention, DNA fragments are generated which direct the incorporation of encoded polypeptides into peroxisomes. Peroxisomes are intracellular cells that are present in large amounts in yeast cells grown in methanol. These intracellular bodies serve to transfer the polypeptide product from intracellular fluids as well as enzymes, e.g. isolated from proteases.

According to another embodiment of the process of the invention, an alcohol oxidase, i.e., a ca. 40 kilolithos4

Genes encoding the production of a protein of 205 627 Β n and a protein of 76 kilodaltons were prepared.

In a further embodiment of the invention, plasmids and transformed organisms containing the DNA fragments described above are prepared.

Culturing host cells transformed with plasmids produced by the method of the invention can produce heterologous polypeptides that were not originally present in the host.

Isolation of regulatory genes from Pichia pastoris

An approx. A 20,000-member cDNA library was constructed in E. coli with polyA ⁺ RNA isolated from Pichia pastoris cells grown on metal as the sole carbon source (see Example III). The library was screened by hybridization using kinase-treated polyA ⁺ RNA isolated from Pichia pastoris grown in methanol or ethanol. After several such minus screenings, three distinct non-homologous cDNA clones were identified as copies of the methanol-specific messenger RNAs. These clones are designated pPC 6.4 pPC 8.0 and pPC 15.0 and are determined to contain inserts of 470, 750, and 1100 nucleotides in length.

In an experiment to obtain larger length cDNA clones, a second cDNA library was constructed using milder S1 nuclease digestion conditions than used for the first cDNA library, and members of this new library were screened individually with ³² P-labeled pPC 6.4. , pPC 8.0 and pPC 15.0 cDNA clones. As a result, larger cDNA clones corresponding to pPC 6.4 and pPC 8.0 cDNA clones were isolated. Larger clones pPC 6.7 and pPC 8.3 are found to contain inserts of 1200 and 2100 nucleotides respectively (see Figures 2 and 3). No cDNA clone having an insert greater than 1100 nucleotides of pPC 15.0 was observed when screening more than 40,000 cDNA clones.

Isolation of DNA fragments from the corresponding genome of each of these DNA clones is accomplished by digestion of the Picha pastoris restriction endonuclease-treated chromosomal DNA fragments hybridizing with ³² P-labeled pPC 15.0, pPC 8.0, or pPC 6.4, and by electroelution with agarose. . The eluted genomic DNA fragments are then cloned into Escherichia coli and the corresponding genomic clones identified by multiple screening with each of the above cDNA probing samples.

The relationship between each cDNA clone and its corresponding genomic clone is depicted in Figures 1, 2 and 3. PPC 15.0 is encoded within a 6,000 nucleotide HindIII genomic fragment that is present in pPG 6.0 clone (Figure 1). The 5 'end of the gene encoded by pPC 15.0 is directed towards the 1300 bp HindIII-EcoRI fragment in pPG 6.0, while the 3' end of the gene is in close proximity to the PstI sites in pPG 6.0.

The pPC 8.3 cDNA clone is included in the pPG 4.0 genomic clone (Figure 2). PPG 4.0 includes the insertion of 4,000 nucleotides of EcoRI-PvuII effective genomic DNA. The orientation of pPC 8.3 within pPG 4.0 places the 5 'end of the gene for this cDNA clone close to the BamHI sites, while the 3' end of this gene is located near the PvuII site. The orientation of pPC 8.0 (a related cDNA clone) within pPG 4.0 places the 5 'end of this cDNA clone near the KpnI site at the 3' end of pPG 4.0, and the 4 'end of the cDNA clone near the PvuII site is placed.

The pPC 6.7 cDNA clone is located completely within a EcoRI-BamHI genomic fragment containing 4800 nucleotides (see Figure 3). The pPC 6.4 clone, on the other hand, is completely within the pPC 6.7 cDNA clone. Since pPC 6.7 is a much more complete copy than pPC 6.4, the latter is not considered further. The 5 'end of the gene is located closer to the BamHI end than the EcoRI end of the pPG 4.8 genomic clone (Figure 3).

Each of the genomic clones described above contains at least 1.2 kilobase pairs of flanking genomic DNA sequences located in the 5 'direction relative to the copied structural genes in each cDNA clone.

The genomic and cDNA clones described above were deposited with E. coli hosts at the United States Northern Region Research Center (NRRL, Peoria, Illinois) on August 31, 1984:

plasmid host Deposit number pPG 6.0 E. coli LE392-pPG 6.0 NRRLB-15 867 pPg 4.0 E. coli LE392-pPG 4.0 NRRLB-15,868 pPg 4.8 E. coli LE392-pPG 4.8 NRRL B-15 869 pPC 15.0 E. coli LE392-pPG 15.0 NRRLB-15,870 pPC 8.3 E. coli LE392-pPC 8.3 NRRLB-15 871 pPC 6.7 E. coli LE392-pPC 6.7 NRRLB-15 872 pPC 8.0 E. coli LE392-pPC 8.0 NRRL B-15 873

Unique nature of pPG 6.0, pPG 4.0, and pPG 4.8 in methanol assimilating yeasts Each of the cDNA clones described above is labeled as chromosomal DNA sequences from a variety of methanol-assimilating yeast and non-methanol assimilating yeast. Homologous genes in each of the three cDNAs are found to exist in substantially all of the methanol-assimilating yeasts but are clearly absent in non-methanol-assimilating yeasts (S. cerevisiae). Therefore, we believe that these genes are unique in methanol-assimilating yeasts. In addition, the XVH. Examples of Southern hybridization experiments detailed in Example 1A show that there is a high degree of homology between the individual methanol-sensitive genes obtained from different methanol-assimilating yeasts.

Characterization of RNA transcripts of the pPG 6.0, pPG 4.0 and pPG 4.8 genes

The influence of methanol can be observed on this clone5

EN 205,627 by studying the effects of these genes on transcription. PolyA ⁺ RNA isolated from Pichia pastoris cells grown on ethanol or methanol as a unique carbon source was used to screen for three identical filter pairs (from methanol and ethanol grown cells) to produce Northern hybridization filters (see Example IV). Example 1) with ³² P-labeled pPC 15.0, pPC 8.0 and pPC 6.4, respectively. Clones pPC 15.0, pPC 8.0 and pPC 6.4 hybridize to RNA molecules (approximately 2400,2300 and 1300 nucleotides in length) from cells grown in methanol, respectively. Hybridization of the pPC 15.0 and pPC 8.0 clones was not observed with RNA hybridization probes obtained from cells grown in ethanol. However, when RNA isolated from cells grown in ethanol is probed with pPC 6.4, the clone hybridizes to a 1300 nucleotide RNA molecule identical to that seen with methanol grown cells, but at a (qualitatively) fivefold lower level.

Determine the size of protein products encoded by pPG 6.0, pPG 4.0 and pPG 4.8

In order to determine which protein products are encoded by the cDNA clones identified above, polyA ⁺ RNA obtained from methanol-grown Pichia pastoris cells is selectively hybridized to each cDNA clone. The hybrid-selected mRNA, i.e., the mRNA which hybridizes to each cDNA clone, is then used for in vitro translation and each protein product is recovered by electrophoresis under the conditions of SDS denaturation (see Example V). The results of these in vitro positive hybridization translational experiments indicate that clones pPC 15.0, pPC 8.3 and pPC 6.7 select mRNAs that are 76,000 (p76), 72,000 (p72), and 40,000 (p40). ) encodes information for dalton molecular weight polypeptides. The same proteins are observed when total polyA ⁺ RNA (i.e., non-hybrid selected RNA) from Pichia pastoris cells grown on methanol is translated into the same in vitro system.

Identification of p72 as an alcohol oxidase

A. Molecular Weight Comparison A highly enriched sample in alcohol oxidase is prepared by dialysis of clarified cell lysates against water (see Example VH). The crystalline precipitate obtained from this dialysis was found to contain two polypeptides with a molecular weight of 76,000 and 72,000 daltons, respectively, by SDS electrophoresis. The precipitate was further purified by chromatography on Sephacryl 200 (see Example VII) demonstrating that the alcohol oxidase activity corresponds to that of the purified 72,000 Dalton polypeptide. The size of this polypeptide is the same as the size of the protein product selected by the pPC 8.3 cDNA clone (see Example X).

B) Immune strike

The cloning of the alcohol oxidase gene by clones pPC 8.3 and pPG 4.0 is also confirmed immunologically (Example XI). Protein preparations isolated from Pichia pastoris containing both the 76,000 and 72,000 molecular weight polypeptides produce antisera specific for these polypeptides in rabbits. When hybrid selected polyA ⁺ RNA from pPC 8.3 is subjected to in vitro translation, only the translation product of 72,000 Dalton molecular weight precipitates with antiserum to protein preparations from Pichia pastoris cells.

C) Comparison of Predicted Amino Acid Sequences

To further confirm that pPC clone 8.3 is indeed the cDNA clone encoding Pichia pastoris alcohol oxidase, the amino acid sequence at the amino terminus of the protein is compared to the predicted amino acid sequence of pPC 8.3. Thus, the NH ₂ -terminal amino acid sequence (SEQ ID NO: A) of the isolated protein of 72,000 daltons was determined as follows (Example VIH): Ala-He-Pro-Glu-Glu-Phe-Asp-He-Leu-Val-Leu -GIyGly-Gly-Ser-Ser-Gly-Ser.

Sequence A

In parallel, the nucleotide sequence of the 5 'end of the genes encoded in pPC 8.3 and pPG 4.0 was determined. The predicted amino acid sequence for the 2-19 amino acids derived from the DNA sequences of both the genomic and cDNA clones (see SEQ ID NO: B) perfectly matched the first 18 amino acids of the above Pichia pastoris alcohol oxidase sequence (SEQ ID NO: A):

Predicted amino acid sequence:

Met Alá Ile Pro Glu Glu Phe

Nucleotide sequence

5 '-ATG GCT ATC CCC GAA GAG TTT (pPC 8.3 and pPG 4.0)

3 '-TAC CGA TAG GGG CTT CTC AAA

Asp Ile Leu Val Leu Gly Gly Gly Ser Ser Gly Ser GAT ATC CTA GTT CTA GGT GGT GGA TCC AGT GGA TCC-3 'CTA TAS GAT CAA GAT CCA CCA CCT AGG TCA CCT AGG-115' Sequence B

In addition, the complete nucleotide sequence belonging to the gene coding region of the alcohol oxidase is determined. The determined nucleotide sequence and the predicted amino acid sequence are as follows: Predicted amino acid sequence: Met Alá Ile Pro Glu Glu Phe

nucleotide sequence:

5 '-ATG GCT ATC CCC GAA GAG TTT (pPC 8.3 and pPG 4.0)

3 '-TAC CGA TAG GGG CTT CTC AAA

Asp Ile Leu Val Leu Gly Gly Gly Ser Ser Gly Ser GAT ATC CTA GTT CTA GGT GGT GGA TTC AGT GGA TCC

CTA TAG GAT CAA GAT CCA CCA CCT AGG TCA CCT AGG

Cys Ile Ser Gly Arg Leu Al Asn Leu Asp His Ser TGT ATT TCC GGA AGA TTG GCA AAC TTG GAC CAC TCC

ACA TAA AGG CCT TCT AAC CGT TTG AAC CTG GTG AGG

HU

Leu Lys Val Gly Leu Ile Glu Alá Gly Glu Asn Gin TTG AAA GTT GGT CTT ATC GAA GCA GGT GAG AAC CAA AAC TTT CAA CCA GAA TAG CTT CGT CCA CTC TTG GTT

Pro Gin Gin Pro Met Gly Leu Pro Ser Arg Tyr Leu

CCT CAA CAA CCC ATG GGT CTA CCT TCC AGG TAT TTA

GGA GTT GTT GGG TAC CCA GAT GGA AGG TCC ATA AAT

Pro Lys Lys Gin Lys Leu Asp Ser Lys Thr Alá Ser

CCC AAG AAA CAG AAG TTG GAC TTC AAG ACT GCT TCC

GGG TTC TTT GTC TTC AAC CTG AGG TTC TGA CGA AGG

Phe Tyr Thr Ser Asn Pro Ser Pro His Leu Asn Gly

TTC TAC ACT TCT AAC CCA TCT CCT CAC TTG AAT GGT

AAG ATG TGA AGA TTG GGT AGA GGA GTG AAC TTA CCA

Arg Arg Alá Ile Val Pro Cys Alá Asn Val Leu Gly

AGA AGA GCC ATC GTT CCA TGT GCT AAC GTC TTG GGT

TCT TCT CGG TAG CAA GGT ACA CGA TTG CAG AAC CCA

Gly Gly Ser Ser Ile Asn Phe Met Met Tyr Thr Arg

GGT GGT TCT TCT ATC AAC TTC ATG ATG TAC ACC AGA

CCA CCA AGA AGA TAG TTG AAG TAC TAC ATG TGG TCT

Gly Ser Alá Ser Asp Ser Asp Asp? Gin Alá Glu

GGT TCT GCT TCT GAT TCT GAT GAC TTN CAA GCC GAG

CCA AGA CGA AGA CTA AGA CTA CTG AAN GTT CGG CTC

Gly Ser Lys Thr Glu Asp Leu Leu Pro Leu Met Lys

GGC TCG AAA ACA GAG GAC TTG CTT CCA TTG ATG AAA

CCG AGC TTT TGT CTC CTG AAC GAA GGT AAC TAC TTT

Lys Thr Glu Thr Tyr Gin Arg Alá? Gin? Tyr

AAG ACT GAG ACC TAC CAA AGA GCT TGN CAA CNA TAC

TTC TGA CTC TGG ATG GTT TCT CGA ACN GTT GNT ATG

Pro Asp Ile His Gly Phe Glu Gly Pro Ile Lys Val

CCT GAC ATT CAC GGT TTC GAA GGT CCA ATC AAG GTT

GGA CTG TAA GTG CCA AAG CTT CCA GGT TAG TTC CAA

Ser Phe Gly Asn Tyr Thr Tyr Pro Val Cys Gin Asp

TCT TTC GGT AAC TAC ACC TAC CCA GTT TGC CAG GAC

AGA AAG CCA TTG ATG TGG ATG GGT CAA ACG GTC CTG

Phe Leu Arg Alá Ser Glu Ser Gin Gly Ile Pro Tyr

TTC TTG AGG GCT TCT GAG TCC CAA GGT ATT CCA TAC

AAG AAC TCC CGA AGA CTC AGG GTT CCA TAA CGT ATG

Val Asp Asp Leu Glu Asp Leu Val Leu Thr His Gly

GTT GAC GAT CTG GAA GAC TTG GTA CTG ACT CAC GGT

CAA CTG CTA GAC CTT CTG AAC CAT GAC TGA GTG CCA

Alá Glu His Trp Leu Lys Trp Ile Asn Arg Asp Thr

GCT GAA CAC TGG TTG AAG TGG ATC AAC AGA GAC ACT

CGA CTT GTG ACC AAC TTC ACC TAG TTC TCT CTG TGA

Gly Arg Arg Ser Asp Ser Alá His Alá Phe Val His

CGT CGT TCC GAC TCT GCT CAT GCA TTT GTC CAC TCT

GCA GCA AGG CTG AGA CGA GTA CGT AAA CAG GTG AGA

Ser Thr Met Arg Asn His Asp Asn Leu Tyr Leu Ile

TCT ACT ATG AGA AAC CAC GAC AAC TTG TAC TTG ATG

AGA TGA TAC TCT TTG GTG CTG TTG AAC ATG AAC TAG

Cys Asn Thr Lys Val Asp Lys Ile Ile Val Glu Asp

TGT AAC ACG AAG GTC GAC AAA ATT ATT GTC GAA GAC

ACA TTG TGC TTC CAG CTG TTT TAA TAA CAG CTT CTG

Gly Arg Alá Alá Alá Val Arg Thr Val Pro Ser Lys

GGA AGA GCT GCT GCT GTT AGA ACC GTT CCA AGC AAG

CCT TCT CGA CGA CGA CAA TCT TGG CAA GGT TCG TTC

Pro Leu Asn Pro Lys Lys Pro Ser Hiy Lys Ile Tyr

CCT TTG AAC CCA AAG AAG CCA AGT CAC AAG ATC TAC

GCA AAC TTG GGT TTC TTC GGT TCA GTG TTC TAG ATG

Arg Alá Arg Lys Gin Ile Phe Leu Ser Cys Gly Thr

CGT GCT AGA AAG CAA ATC TTT TTG TCT TGT GGT ACC

GCA CGA TCT TTC GTT TAG AAA AAC AGA ACA CCA TGG

627 B

Ile Ser Ser Pro Leu Val Leu Glu Arg Ser Gly Phe

ATC TCC TCT CCA TTG GTT TTG CAA AGA TCC GGT TTT

TAG AGG AGA GGT AAC CAA AAC GTT TCT AGG CCA AAA

Gly Asp Pro Ile Lys Leu Arg Under Al Gly Val Lys

GGT GAC CCA ATC AAG TTG AGA GCC GCT GGT GTT AAG

CCA CTG GGT TAG TTC AAC TCT CGG CGA CCA CAA TTC

Pro Leu Val Asn Leu Pro Gly Val Gly Arg Asn Phe

CCT TTG GTC AAC TTG CCA GGT GTC GGA AGA AAC TTC

GGA AAC CAG TTG AAC GGT CCA CAG CCT TCT TTG AAG

Gly Asp His Thr Lys Ile Pro Pro Gly Lys Tyr Met

GGT GAC CAC ACC AAG ATT CCT CCT GGA AAG TAC ATG

CCA CTG GTG TGG TTC TAA GGA GGA CCT TTC ATG TAC

Thr Met Phe His Phe Leu Glu Tyr Pro Phe Ser Arg

ACT ATG TTC CAC TTC TTG GAA TAC CCA TTC TCC AGA

TGA TAC AAG GTG AAG AAC CTT ATG GGT AAG AGG TCT

Gly Ser Ile His Ile Thr Ser Pro Asp Pro Tyr Alá

CGT TCC ATT CAC ATT ACC TCC CCA GAC CCA TAC GCA

CCA AGG TAA GTG TAA TGG AGG GGT CTG GGT ATG CGT

Alá Pro Asp Phe Asp Arg Gly Phe Met Asn Asp Glu

GCT CCA GAC TTC GAC CGA GGT TTC ATG AAC GAT GAA

CGA GGT CTG AAG CTG GCT CCA AAG TAC TTG CTA CTT

Arg Asp Met Alá Pro Met Val Trp Al Tyr Tyr Lys Ser

AGA GAC ATG GCT CCT ATG GTT TGG GCT TAC AAG TCT

TCT CTG TAC CGA GGA TAC CAA ACC CGA ATG TTC AGA

Ser Arg Glu Thr Alá Arg Arg Ser Asp His Phe Alá

TCT AGA GAA ACC GCT AGA AGA AGT GAC CAG TTT GCC

AGA TCT CTT TGG CGA TCT TCT TCA CTG GTG AAA CGG

Gly Glu Val Thr Ser His His Pro Leu Phe Pro Tyr

GGT GAG GTC ACT TCT CAC CAC CCT CTG TTC CCA TAC

CCA CTC CAG TGA AGA GTG GTG GGA GAC AAG GGT ATG

Ser Ser Glu Alá Arg Alá Leu Glu Met Asp Leu Glu

TCA TCC GAG GCC AGA GCC TTG GAA ATG GAT TTG GAG

AGT AGG CTC CGG TCT CGG AAC CTT TAC CTA AAC CTC

Thr Ser Asn Alá Tyr Gly Gly Pro Leu Asn Leu Ser

ACC TCT AAT GCC TAC GGT GGA CCT TTG AAC TTG TCT

TGG AGA TTA CGG ATG CCA CCT GGA AAC TTG AAC AGA

Alá Gly Leu Alá His Gly Ser Trp Thr Gin Pro Leu

GCT GGT CTT GCT CAC GGT TCT TGG ACT CAA CCT TTG

CGA CCA GAA CGA GTG CCA AGA ACC TGA GTT GGA AAC

Lys Lys Pro Thr Alá Lys Asn Glu Gly His Val Thr

AAG AAG CCA ACT GCA AAG AAC GAA GGC CAC GIT ACT

TTC TTC GGT TGA CGT TTC TTG CTT CCG GTG CAA TGA

Ser Asn Gin Val Glu Leu His Pro Asp Ile Glu Tyr

TCG AAC CAG GTC GAG CTT CAT CCA GAC ATC GAG TAC

AGC TTG GTC CAG CTC GAA GTA GGT CTG TAG CTC ATG

Asp Glu Glu Asp Asp Lys Alá Ile Glu Asn Tyr Ile

GAT GAG GAG GAT GAC AAG GCC ATT GAG ACC TAC ATT

CTA CTC CTC CTA CTG TTC CGG TAA CTC TTG ATG TAA

Arg Glu His Thr Glu Thr Thr Trp His Cys Leu Gly

CGT GAG CAC ACT GAG ACC ACA TGG CAC TGT CTG GGA

GCA CTC GTG TGA CTC TGG TGT ACC GTG ACA CCA GGT

Thr Cys Ser Ile Gly Pro Arg Glu Gly Ser Lys Ile

ACC TGT TCC ATC GGT CCA AGA GAA GGT TCC AAG ATC

TGG ACA AGG TAG CCA GGT TCT CTT CCA AGG TTC TAG

Val Lys Trp Gly Gly Val Leu Asp His Arg Ser Asn

GTC AAA TGG GGT GTT GTT TIG GAC CAC AGA TCC AAC

CAG TTT ACC CCA CAA CAA AAC CTG GTG TCT AGG TTG

Val Tyr Gly Val Lys Gly Leu Lys Val Gly Asp Leu

GTT TAC GGA GTC AAG GGC TIG AAG GTT GGT GAC TTG

CAA ATG CCT CAG TTC CCG AAC TTC CAA CCA CTG AAC

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Ser Val Cys Pro Asp Asn Val Gly Cys Asn Thr Tyr TCC GTG TGC CCA GAC AAT GTT GGT TGT AAC ACC TAC AGG CAC ACG GGT CTG TTA CAA CCA ACA TTG TGG ATG

Thr Thr Alá Leu Leu Ile Gly Glu Lys Thr Alá Thr ACC ACC GCT CTT TTG ATC GGT GAA AAG ACT GCC ACT TGG TGG CGA GAA AAC TAG CCA CTT TTC TGA CGG TGA

Leu Val Gly Glu Asp Leu Gly Tyr Ser Gly Glu Alá TTG GTT GGA GAA CAT TTA GGA TAC TCT GGT GAG GCC AAC CAA CCT CTT CTA AAT CCT ATG AGA CCA CTC CGG

Leu Asp Met Thr Val Pro Gin Phe Lys Leu Gly Thr TTA GAC ATG ACT GTT CCT CAG TTC AAG TTG GGC ACT AAT CTG TAC TGA CAA GGA GTC AAG TTC AAC CCG TGA

Tyr Glu Lys Thr Gly Leu Alá Arg Phe Stop

TAC GAG AAG ACC GGT CTT GCT AGA TTC TAA-3 '

ATG CTC TTC TGG CCA GAA CGA TCT AAG ATT-5 '

Sequence B '

Comparison of the above nucleotide sequence with that of the previously described alcohol oxidase from Hansenula polymorphus published by Ledeboer et al. Reveals several significant differences, including the predicted amino acid sequence, the actual size of the gene (and the resulting protein), codon deletion hair.

Identification of p76 as dihydroxyacetone synthase

For the first 51 nucleotides of the p76 gene, the nucleotide sequence is determined by standard techniques. From this sequence, the amino acid sequence of the amino terminus of the p76 protein can be predicted:

Amino acid sequence: Met Alá Arg Ile Pro Lys

nucleotide sequence:

5 '-ATG GCT AGA ATT CCA AAA

3 '-TAC CGA TCT TAA GGT TTT

Pro Val Ser Thr Gin Asp Asp Ile His Gly Leu

CCA GTA TCG ACA CAA GAT GAC ATT CAT GAA TTG-3 '

GGT CAT AGC TGT GTT CTA CTG TAA GTA CTT AAC-5 '

This predicted amino acid sequence of p76 can be compared to the published amino acid sequence of the dihydroxyacetone synthase (DHAS) protein from Hansenula polymorph (Manowi et al.). Although there are many differences between the two sequences, there are similarities between the two proteins that can be recognized:

PichiaDHAS:

Met-Ala -Arg-Ile-Pro-Lys-ProHansenula DHAS:

Met-Ser-Met-Arg-Ile-Pro-Lys -AlaVal-Ser-Thr-Gln-Asp-Ile-His-Glu-Leu.Ala-Ser-Val-Asn-Asp-Glu-Gln-His-Gln -Axg-IleA Based on the significant homology of Pichia p76 and Hansenula DHAS and based on a similar protein size (about 76,000 Daltons), p76 can be tentatively identified as Pichia DHAS.

As discussed above for the alcohol oxidase gene, comparison of the nucleotide sequence of the first 51 nucleotides of the Pichia DHAS protein with that of Hansenula DHAS previously published (Janowicz et al.) Suggests a number of differences in codon displacement, predicted amino acid sequence, etc.

DNA fragments containing Pichia pastoris containing regulatory promoters

The regulatory regions 5 'produced by the method of the invention are detailed in the restriction maps shown in Figures 4, 5 and 6. The regulatory region 5 ', which regulates expression of the p76 polypeptide, is located within the DNA fragment shown in Figure 4a. The HindHl kil / ζοΙ fragment of about 2.9 kilobase pairs was clearly shown to contain the regulatory region, as further elaborated below.

Because the cDNA clone pPC 15.0 is not a complete cDNA copy, it is most likely that at least a portion of the DNA fragment depicted in Figure 4a contains structural coding sequences for the p76 polypeptide. Thus, the regulatory function is believed to be present in the approximately 1300 base pair HindIII-EcoRI fragment shown in Figure 4b. Structures containing the novel β-galactosidase gene, which will be discussed in more detail below, support this view.

The regulatory region 5 ', which regulates the expression of the p72 polypeptide (alcohol oxidase), is located within the approximately 2000 base pair EcoRI-EcoRV DNA fragment shown in Figure 5. The novel β-galactosidase gene-containing constructs described below demonstrate the controllable nature of this DNA fragment.

Figure 6 shows a restriction map of a BamHI-SalI DNA fragment of about 3 kilobase pairs comprising the 5 'regulatory region regulating p40 polypeptide production. This fragment is clearly distinguished from the 5 'regulatory regions detailed in FIGS. 4 and 5, including by the different restriction sites within the DNA fragment.

Figures A10, 2a and 11 show restriction enzyme data for regulatory domains and structural genes for the p76, p72 (alcohol oxidase) and p40 polypeptides, respectively. In particular, Figure 10 shows details of a 3.8 kilobase HindIII-PII fragment of Pichia pastoris genomic DNA that regulates and encodes the production of the p76 polypeptide. Figure 2a is a 4.0 kb EcoRI-PvuH fragment of Pichia pastoris genomic DNA that regulates and encodes the production of the p72 (alcohol oxidase) polypeptide. II. Figure 3B shows a 3.7 kb BamHI-EcoRI fragment of Pichia pastoris genomic DNA that regulates and encodes the production of the p4β polypeptide.

Genomic clones pPG 6.0, pPG 4.0 and pPG 4.8 can also be characterized by restriction mapping. Thus, pPG 6.0 clone is detailed in Figure 1a). As a point of orientation, the 5 'end of the DNA fragment is considered to be the origin G, origin'). PPG 6.0 is a HindIII fragment of the Pichia pastoris chromosomal DNA, which is about 6 kilobase pairs in length and is cleaved by the following different restriction enzymes:

HU 205 627 Β

restriction enzyme rip places Distance from origin bp Hinc 5 1070,1740,1890,3320, 5520 Eco 2 1300.3450 Xho 1 2860 Pst Second 3820.4200 PvuII 1 4120 PvuI 1 4950

The pPg 4.0 clone is shown in detail in Figure 2a. The clone is an EcoRI-HindIII fragment of chromosomal DNA about 4 kilobase pairs in length. Considering the origin of the 5 'end of the clone, the following restriction data for pPG 4.0 are obtained:

Restriction enzyme Cleavage sites Distance from the origin (Origin) bp Hind III 3 400,600,1840 Pst 1 8500 Bam 2 1960.1970 Sáli 1 2620 BglII 2 1040.2700 KpnI 2 500, 2730 XbaI 1 3330 Stu 1 3880 Nde 1 420 Hindi 2 870.2430 Sst 1 1200 Bell 2 1710, 4080 AsuH 2 1900.2300 RV 1 1930 Pvull 1 4120

Clone pPG 4.8 is depicted in detail in Figure 3a. The clone is a 4.8 kilobase BamHI-EcoRI fragment of Pichia pastoris chromosomal DNA, which has the following additional restriction sites:

Restriction enzyme Cleavage sites Distance from origin bp Cla 1 410 KpnI 3 500,3890.4280 PvuI 1 1120 Sáli 1 2900 Pvull 1 4135 EcoRV 2 3690, 3890 BglII 1 4500 XmaI 1 4800

The genomic clones pPG 6.0, pPG 4.0 and pPG 4.8 were manipulated by inserting into unique restriction sites on E. coli plasmid pBR322. Clone pPG 6.0, a HindIII fragment, was simply cloned into the HindIII site of pBR322. Clone pPG 4.0 was cloned into the EcoRI-PvuU sites of pBR322 and pPG 4.8 was cloned into the EcoRI-BamHI sites of pBR322 (see Example VI). E. coli strains transformed with these plasmids were deposited at the Northern Region Research Center (NRRL) under the following numbers:

Genome class Lab name Deposit number pPG 6.0 LE392-pPG 6.0 NRRL B-15 867 pPG 4.0 LE392-pPG 4.0 NRRL B-15 868 pPG 4.8 LE392-pPG 4.8 NRRL B-15 869

Figures 7, 8 and 9 show restriction map data for the 3 'regulatory regions of p76, p72 (alcohol oxidase) and p40 polypeptides, respectively. The regulatory regions 3 'are useful in the regulation of polyadenylation, transcription termination and arrest of messenger RNA, which is encoded by the preceding nucleotide sequences. Thus, the 3 'regulatory region derived from the p76 polypeptide gene, a 2.7 kilobase Eco RI-HindIII fragment shown in Figure 7, is useful in regulating polyadenylation, transcription, and translation of mRNA encoding the p76 polypeptide, or any other mRNA. , derived from a gene upstream of the 3 'regulatory region. The 0.2 kilobase pair of StuI-PvuII fragment from p72 gene detailed in Fig. 8a, the 0.3 kilobase pair StuI-HindIII fragment from p72 gene detailed in Fig. 8b, the 3.2 kilobase pair of Sall-EcoRI derived from p72 gene. and the 1.9 kilobase SalI-EcoRI fragment derived from the p40 gene detailed in Figure 9 are similarly useful for the structural genes to which they are linked in wild type Pichia pastoris as for any foreign (i.e. heterologous) genes. that can be inserted upstream of these 3 'control regions.

Because the alcohol oxidase gene in pPG 4.0 is within a few hundred base pairs of the AO gene transcription stop site, the additional 3 'sequence detailed in Figure 8c is obtained as follows. In the first step, the Pichia chromosomal DNA was digested with EcoRI and SalI and the digested DNA was hybridized with the 2.0 kb ³² P-labeled BamHIHindII fragment from the AO gene by Southern blotting. Among the digested fragments of Pichia EcoRI-SalI which hybridize to the AO gene probe is a 3.2 kilobase pair fragment that encodes sequences flanking the 3 'portion of the AO gene and the 3' terminal of the gene.

The 3 'fragment of the AO gene is then cloned by recovering the approximately 3.2 kbp Eco RI-SalI cleaved Pichia DNA fragments by gel elution and inserting them into Eco RI and SalI digested pBR322. Finally, a recombinant plasmid, pPG

3.2, which contains the 3 'fragment of the AO gene, is identified by telephoto hybridization using the AO gene fragment as a probe. An E. coli strain transformed with plasmid pPG 3.2 was deposited with the Northern Region at the Research Center (Peoria, Illinois) under the accession number NRRL B-15999. Figure 8c) is pPG

3.2 shows a restriction endonuclease cleavage site map of the Pichia DNA fragment from 3.2. The fragment encodes a 3 'portion of AO (SalI to HindIII) 1.5

EN 205 627 Β kbp and about 1.7 kbp of the 3 'sequence of the AO gene.

Characterization of cDNA clones

CDNA clones belonging to the regulatory genes from Pichiapastoris were also characterized by restriction mapping. Figure 12 shows a 1.1 kilobase pair fragment of p76 cDNA. Based on the 5 'end of the DNA sequence as the origin, the restriction enzyme XhoI cleaves p76 cDNA at about 500 bp, HincH cleaves at about 950 bp from start, and Eco RI p76 cDNA at start. calculated at about 10501100 base pairs. The cDNA clone shown in Figure 12 and the cDNA clones shown in Figures 13 and 14 are all shown with PstI ends. These are artificially created restriction sites produced by G-C tailing of the originally obtained complementer DNA to facilitate the cloning of the DNA fragments into pBR322. Based on Northern hybridization studies and the size of the polypeptide product, it is estimated that the cDNA clone pPC 15.0 is an imperfect copy of the p76 mRNA, representing only about half of the total messenger RNA sequence.

Figure 13 shows a composite restriction map of p72 (alcohol oxidase) cDNA constructed by overlapping clones pPC 8.3 and pPC 8.0. As above, the 5 'end of the DNA sequence is considered to be "origin". Thus, treatment of the alcohol oxidase cDNA with various restriction enzymes yields fragments of the following size:

Restriction enzyme Cleavage sites Distance from origin bp AsuH 2 20.420 EcoRV 1 50 Bam 2 80,90 Hinch 1 550 Sáli 1 820 BglII 1 820 KpnI 1 850 XbaI 1 1450 RsaI 1 1760 Stu 1 2000

Restriction enzyme mapping of the 3 'end of the alcohol oxidase gene in clones pPC 8.0 and pPC 8.3 reveals that the pPC 8.3 cDNA clone lacks about 250 nucleotides in the alcohol oxidase mRNA sequence (Figure 2). The sequences present at the 3 'end of the alcohol oxidase mRNA are present in the cDNA clone pPC 8.0, which overlaps pPC 8.3 with about 500 nucleotides.

Figure 14 provides a restriction map for the p40 polypeptide cDNA, a 1.2 kilobase pair fragment. Considering the origin of the 5 'end of the cDNA clone, pPC 6.7 is cleaved by SalI (and HincII) by about 1000 bp from origin.

Each cDNA fragment was cloned into pBR322, which was then transformed into E. coli. Transformed strains were deposited at the Northern Region Research Center Institute (Peoria, Illinois) with the following numbers:

cDNA clones Laboratory analysis Deposit number pPC 15.0 pPC 8.3 pPC 8.0 pPC 6.7 LE392.pPC 15.0 LE392-pPC 8.3 MM294_pPC8.0 LE392-pPC 6.7 NRRL B-15,870 NRRL B-15,871 NRRL B-15,873 NRRL B-15,872

Each of the cDNA clones described above is useful as a probe for the identification and isolation of chromosomal DNA encoding the production of unique polypeptides of methanol as the sole carbon and energy source. Therefore, as described previously, these clones are used to identify chromosomal DNA fragments of Pichia pastoris that contain regulatory regions and structural coding information for individual polypeptides that are detected when Pichia pastoris grows on methanol. Similarly, these cDNA clones can be used as probes to identify and recover analogous genes from other methanol-simulating yeasts, e.g. Torulopsis molischiana, Hansenula capsulatum, H. nonfermentans and the like (see Example XVII).

Detailed analysis of the alcohol oxidase gene

The 5 'regulatory region of pPG 4.0 clone is further characterized by determining the nucleotide sequence of the clone "upstream" (5') where the structural information of p72 (alcohol oxidase) is encoded. The first 250 nucleotides upstream of the mRNA translation start site (ATG codon) are believed to be:

5'-ATGCTTCCAA GATTCTGGTG GGAATACIGC TGATAGCCTA

ACGTTCATGA TCAAAATTTA ACTGTTCTAA CCCCTACTTG

GACAGGCAAT ATATAAACAG AAGGAAGCTG CCCTGTCTTA

AACCTTTTTT TTTATCATCA TTATTAGCTT ACTTTCATAA

TTGCGACTGG TTCCAATTGA CAAGCTTTTG ATTTTAACGA

CTTTTAACGA CAACTTGAGA AGATCAAAAA ACAACTAATT ATTCGAAACG-3 '.

C sequence

The promoter function of pPG 4.0 clone is believed to be located within this sequence of nucleotide bases.

In order to further describe this new DNA fragment, an additional 301 nucleotides from the sequence detailed in SEQ ID NO.

Thus, the first 551 nucleotides upstream of the mRNA translation site are believed to be:

5 '-AATGGCCAAAA CTGACAGTTT AAACGCTGTC TTGGAACCTA

ATATGACAAA AGCGTGATCT CATCCAAGAT GAACTAAGTT

TGGTTCGTTG AAATTCTAAC GGCCAGTTGG GAACTAAGTT

ACTTCCAAAA GTCGCCATAC CGTTTGTCTT GTTTGGTATI

GATTGACGAA TGCTCAAAAA TAATCTCATT AATGCTTAGC

GCAGTCTCTC TATCGCTTCT GAACCCGGTG GCACCTGTGC

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CGAAACGCAA ATGGGGAAAC AACCCGCTTT TTGGATGATT

ATGCATTGTC CTCCACATTGT ATGCTTCCAA GATTCTGGTG

GGAATACTGC TGATAGCCTA ACGTTCATGA TCAAAATTTA

ACTGTTCTAA CCCCTACTTG GACAGGCAAT ATATAAACAG

AAGGAAGCTG CCCTGTCTTA AACCTTTTTT TTTATCATCA

TTATTAGCTT ACTTTCATAA TTGCGACTGG TTCCAATTGA

CAAGCTTTTG ATTTTAACGA CTTTTAACGA CAACTTGAGA AGATCAAAAA ACAACTAATT ATTCGAAACG-3 '

D sequence

Additional nucleotides in sequence D (compared to sequence C) are believed to have additional regulatory functions within the C sequence by an unknown mechanism. It can be seen that the sequence D is shown in XIV. and XV. The 1.1 kbp DNA fragment capable of regulating gene expression in yeasts described in Examples 1 to 5 only represents a portion of the DNA sequence. Additional regulatory functions may be encoded within a portion of the 1.1 kbp DNA fragment not detailed in SEQ ID NO: D.

In order to further characterize this novel 1.1 kbp DNA fragment, additional nucleotide sequence analysis was performed to completely outline the XIV. and XV. which is capable of regulating gene expression in yeast. The nucleotide sequence is depicted as the D 'sequence:

5 '-AGATCTAA CATCCAAAGA

CGAAAGGTTG AATGAAACCT TTTTGCCATC CGACATCCAC

AGGTCCATTC TCACACATAA GTGCCAAACG CAACAGGAGG

GGATACACTA GCAGCAGACG TTGCAAACGC AGGACTCATC

CTCTTCTCTA ACACCATTTT GCATGAAAAC AGCCAGTTAT

GGGCTTGATG GAGCTCGCTC ATTCCAATTC CTTCTATTAG

GCTACTAACA CCATGACTTT ATTAGCCTGT CTATCCTGGC

CCCCCTGGCG AGGTCATGTT TGTTTATTTC CGAATGCAAC

AAGCTCCGCA TTACACCCGA ACATCACTCC AGATGAGGGC

TTTCTGAGTG TGGGGTCAAA TAGTTTCATG TTCCCAAATG GCCCAAACT GACAGTTTAA ACGCTGTCTT GGAACCTAAT

ATGACAAAAG CGTGATCTCA TCCAAGATGA ACTAAGTTTG

GTTCGTTGAA ATCCTAACGG CCAGTTGGTC AAAAAGAAAC TTCCAAAAGT CGCCATACCG TTTGTCTTGT TTGGTATTGA TTGACGAATG CTCAAAAATA ATCTCATTAA TGCTTAGCGC AGTCTCTCTA TCGCCTCTGA ACCCGGTGGC ACCTGTGCCG AAACGCAAAT GGGGAAACAA CCCGCTTTTT GGATGATTAT GCATTGTCCT CCACATTGTA TGCTTCCAAG ATTCTGGTGG GAATACTGCT GATAGCCTAA CGTTCATGAT CAAAATTTAA CTGTTCTAAC CCCTACTTGG ACAGGCAATA TATAAACAGA AGGAAGCTGC CCTGTCTTAA ACCTTTTTTT TTATCATCAT TATTAGCTTA CTTTCATAAT TGCGACTGGT TCCAATTGAC AAGCTTTTGA ttttaacgac ttttaacgac aacttgagaa

GATCAAAAAA GAACTAATTA TTCGAAACG-3 '

D '

Those skilled in the art will recognize that additional regulatory functions relative to sequences C and D may be encoded within the portion of sequence D 'further upstream (i.e., 5') from the nucleotide sequences detailed in sequences C and D.

To determine where RNA transcription for the alcohol oxidase gene begins, the DNA sequences around the 5 'end of this gene from the pPG 4.0 genomic clone and the pPC 8.3 cDNA clone are compared. The pPC 8.3 cDNA clone contains about 100 nucleotides of an untranslated region 5 'to the alcohol oxidase gene. Based on this sequence, 15 basic oligonucleotides (5'-CTTCTCAAGTTGTTCG-3 ') were synthesized (see Example IX), which is complementary to nucleotides -29 to -43, where the translational start site (ATG codon) has + 1 nucleotide. and the nucleotide is designated as -1 in the 5 'direction. The 15 basic oligonucleotide is used as a primer to extend along the alcohol oxidase mRNA to reach the 5 'end. The cDNA sequence obtained from this primer extension experiment results in three different transcription start points for Pichia pastoris alcohol oxidase mRNA. The main transcript begins at 144 nucleotides from the translation start codon. Two smaller alternate codons begin at 117 and 111 nucleotides upstream (5 ') from the alcohol oxidase AUG codon.

The 55 nucleotides preceding the start of the alcohol oxidase mRNA contains a putative Goldberg-Hogness "box" (TATAA box).

The TATAAA sequence is located 40 positions upstream of the 5 'end of the predominant transcript for the alcohol oxidase mRNA and therefore 165 nucleotides upstream of the parent codon for this protein.

The 3 'regulatory region of the alcohol oxidase gene is further characterized by determining the nucleotide sequence "downstream" about 120 nucleotides from the point where the structural information for p72 (alcohol oxidase) is encoded. The sequence is shown below as the sequence D ':

5′-TCAAGAGGAT GTCAGAATGC CATTTGCCTG AGAGATGCAG

GTCTCATTTT TGATACTTTT TTATTTGTAA CCTATATAGT

ATAGGATTTT TTTTGTCAAA AAAAAAAAAA AAAAAAAAAA-3 '

D 'sequence

Detailed analysis of the p76 gene

The 5 'regulatory region of pPG 6.0 clone is further characterized by determining the nucleotide sequence of the clone "upstream" (5') where the structural information for p76 is encoded, from the first 622 nucleotides before the mRNA translation start site (ATG codon). we believe that:

5'-TT

CACCCATACA ACTATAAACC TTAGCAÁTTG AAATAACCCC

AATTCATTGT TCCGAGTTTA ATATACTTGC CCCTATAAGA

AACCAAGGGA TTTCAGCTTC CTTACCCCAT GAACAGAATC

TTCCATTTAC CCCCCACTGG AGAGATCCGC CCAAACGAAC

AGATAATAGA AAAAAACAAT TCGGACAAAT AGAACACTTT

CTCAGCCAAT TAAAGTCATT CCATGCACTC CCTTTAGCTG

CCGTTCCATC CCTTTGTTCA GCAACACCAT CGTTAGCCAG

TACGAAAGAG GAAACTTAAC CGATACCTTG GAGAAATCTA

AGGCGCGAAT GAGTTTAGCC TAGATATCCT TAGTGAAGGG

TGTCCGATAC TTCTCCACAT TCAGTCATAG ATGGGCAGCT

TGTATCATGA AGAGACGGAA ACGGGCATAA GGGTAACCGC

HU 205 627 Β

CA & ATTATAT AAAGACAACA TGCCCCAGTT TAAAGTTTTT

CTTTCCTATT CTTGTATCCT GAGTGACCGT TGTGTTTAAT

ATAAAAAGTT CGTTTTAACT TAAGACCAAA ACCAGTTACA

ACAAATTATA ACCCCTCTAA ACACTAAAGT TCACTCTTAT

CAAACTATCA AACATCAAAA-3 '

D '' sequence

The promoter function of clone pPG 6.0 is believed to be within this sequence of nucleotide bases, although those skilled in the art may recognize that additional regulatory properties may result from sequences further upstream of sequences added to sequence D '.

The 3 'regulatory region of clone pPG 6.0 is further characterized by determining the nucleotide sequence "downstream" (in the 3' direction) about 180 nucleotides from the point where the structural information of p76 is encoded. The sequence is set forth below as the D '' sequence

5 '-GTCAGCAGTG TTTCCTGCCA AAGCCATCAA GAGGACGTAC

ATGCTCTCAT TTTTTGGTTT TCTATGTCCG ACGGGGTTCG

TAAACTGGCT TCCTCCTTTT CTTTTCCTGT TGCATTTTAT

TTGGTCAAAC AAAACTAGGG TCTTTTCCTA AAACCTTATG

TCAATGGACC TACCACATAG-3 '

D '' 'sequence

Expression of transformed yeast

The above-described plasmids of the present invention are useful in yeasts which are transformable. The regulation of gene expression in yeast can be accomplished by the novel DNA fragments of the present invention by subjecting the transformed organisms to carbon spinning. Carbon source starvation and induction of expression of a gene product under control of the regulatory sequences of the invention after growth on various catabolite repressing and non-catabolite repressing carbon sources. Another way to achieve expression of desired gene products in appropriate species of transformed yeast is to grow the transformed yeast in methanol. Another way to reduce expression of the desired gene product is to grow the transformed yeast on a medium containing non-catabolite repressing carbon sources.

The regulatory regions of the present invention are useful for expression in all yeast strains since the regulatory regions have been shown to be inducible under various conditions. so e.g. yeasts capable of growing on methanolic or non-catabolite-repressing carbon sources can be induced to produce foreign, i.e., heterologous, olipeptides; whereas yeasts capable of growing on catabolite repressing carbon sources can be induced to produce foreign polypeptide by subjecting the yeast cells thus grown to carbon source starvation conditions.

Preferred members of the following genera for transformation in the method of the invention are:

Candida,

Kloeckera,

Saccharomyces,

Schizosaccharomyces,

Rhodotorula,

Hansenula,

Torulopsis,

Pichia and

K..

Yeasts derived from these nationalities are advantageous because their treatment is safe, their growth and other conditions are well defined and well known to those skilled in the art.

Especially preferred yeast strains for use in one embodiment of the method of the present invention are those yeast strains that are capable of growing on methanol as the sole source of carbon and energy. Yeasts known to be capable of growing on methanol are members of the following nationalities:

Candida,

Kloeckera,

Saccharomyces,

Rhodotorula,

Hansenula,

Torulopsis and

Pichia.

Because the regulatory regions of the present invention may also be induced by growth in non-catabolite repressing carbon sources and under carbon source starvation conditions, yeast strains capable of growing on non-methanol substrates such as:

glucose, acetate, glycerol, ethanol, lactose, galactose, fructose, sucrose and the like, and mixtures of two or more of the foregoing may also be used. With host growth, a suitable non-catabolite repressing non-methanol carbon source, e.g. glycerol and galactose; or, by host growth, a suitable catabolite repressing carbon source, e.g. ethanol, glucose and fructose, and then exposing the host to carbon source starvation conditions, expression of the gene product can be achieved under the control of the regulatory regions of the invention.

In addition, since the regulatory regions of the invention are sensitive to different growth conditions in terms of both induction and repression of expression, regulated expression of the gene product can be achieved under the control of the regulatory regions of the invention. so e.g. cells can be grown on a carbon source that only induces foreign gene expression at a low level and then switched to methanol, which is then strongly induced by gene expression12.

EN 205 627 Β to prevent damage. Alternatively, gene expression may be achieved by mixing inducing / repressing nutrients, e.g. methanol-glucose mixture. In another method, high levels of expression induced by growth in methanol can be reduced if necessary by repressing a carbon source, e.g. by adding glucose or ethanol to the growth medium. Of course, those of ordinary skill in the art will recognize that other variations of nutrient mixtures and nutrient delivery sequences are possible and offer great potential for regulation at the level of gene expression derived from the regulatory domains of the invention.

A particularly preferred yeast strain is Pichia pastoris GS115, which is a mutant deficient in histidine production and thus has a His4 designation by its mutant genotype. GS115 is from Pichia pastoris NRRL Y-ll 430 and is deposited with the Northern Territory Research Center (Peoria, Illinois) of the United States Department of Agriculture, Northern Region. Pichia pastoris GS115 is deposited under accession number NRRL Y-15 851. This host is useful because it is a histidine deficient auxotrophic mutant. Transformation of this host with a vector which also contains the HIS4 gene function among other DNA sequences allows for easy selection of the transformed host.

The regulatory domains of the present invention have also been shown to be useful for the regulated expression of heterologous gene products in the yeast strains of the Saccharomyces nationality, many yeast auxotrophic mutants being known in the yeast. Other preferred host yeast strains include:

ATCC 24,683 (trp 1, ade 1, his 2, leu 1, gal 1, lord 1 mutant), ATCC 24,684 (trp 1, ade 1, his 7, gal 1, lord 1 mutant), ATCC 32,810 (tpr 5, arg 4, his 5, lys 1, ade 2, gal 2 mutant), ATCC 34 182 (ade 3, his, lys, ura mutant), ATCC 34 352 (ura 2 mutant), ATCC 34 353 (ura 2 mutant) ), ATCC 34 353 (lord 2 mutant), ATCC 38 523 (arg 1, thr 1 mutant), ATCC 38 626 (leu 2, his 4 mutant), ATCC 38 660 (his 4, leu 2, thr 4 mutant), ATCC 42 243 (lord 3 mutant), ATCC 42 336 (ade 1, his 4, thr 4 mutant), ATCC 42 403 (arg 4, lys 7 mutant), ATCC 42 404 (ade 1, his 4, leu 2 mutant) , ATCC 42 564 (lord 1, his 6 mutant), ATCC 42 596 (his 4, leu 2, lys 1 mutant), ATCC 42 957 (his 4, leu 2, thr 4, trp 5 mutant), ATCC 42 950 ( ade mutant), ATCC 42,951 (ade, leu mutant), ATCC 44,069 (lord 1 mutant), ATCC 44070 (leu 2, his 4 mutant), ATCC 44,222 (his 4 mutant), ATCC 44,376 (his 4, ade 2 mutant), ATCC 44,377 (his 4, leu 1 mutant) and the like, these are readily available to those who skilled in the art.

Those skilled in the art will recognize that useful host strains are not limited to auxotrophic mutants. Thus PROT with positive selection markers, e ', antibiotic resistance ^genes: ROF strains transformation also provides a useful means for the detection and isolation of transformed strains.

Escherichia coli is also a suitable host for the plasmids of the invention. Those skilled in the art will recognize that many E. coli strains are suitable hosts. The different strains used in this work are summarized in the following table:

Strain Indication Deposit number

MC 1061 is not known

LE392 ATCC 33,572

MM294 'ATCC 33,625

Transformation of Pichia pastoris

Transformation of Pichia pastoris has not been described before. The experimental procedures for transforming Pichia pastoris are described in greater detail later (Example XII). In order to develop a transformation system for P. pastoris, the GS115 auxotrophic mutant (NRRL Y-15851) was isolated and determined to be deficient in the histidine, i.e., lacking detectable histidine dehydrogenase activity.

GS115 (NRRL Y-15,851) can be transformed by enzymatic digestion of cell walls to yield spheroplasts; the spheroplasts are then mixed with the transforming DNA and incubated in the presence of calcium ions and polyethylene glycol and regenerated in selective growing medium with histidine deficiency. The transforming gene includes the HIS4 gene, in which the host is deficient, so that only the transformed cells survive growth on the selective medium used.

Isolation of the Pichia pastoris HIS4 gene

The HIS4 gene was isolated from P. pastoris NRRL Y-113030 by partial digestion of total chromosomal DNA with Sau3A followed by centrifugation on a sucrose gradient (see Example XIII). The 5-20 kbp fragments are cloned into the BamHI site of the S. cerevisiae E. coli shuttle vector YEpl3 (ATCC 37,115; Fig. 33) and transformed into E. coli. About 50,000 colonies were pooled and total plasmid DNA was extracted. Spheroplasts of S. cerevisiae strain 5799-4D (NRRL Y-15,859), a mutant of his4ABC, were mixed with about 1 μg of the YEpl3 Pichia DNA "library" according to Hinnen et al. (1978) and allowed to regenerate in media lacking histidine. . Transformation results in approximately 1x10 ³ prototrophic yeast colonies out of a total population of 5x10 ⁷ regenerable spheroplasts. A duplicate control sample does not produce colonies when incubated without DNA. From 20 of His ⁺ colonies, the whole yeast DNA was extracted and transformed into E. coli. Of the yeast DNA preparations, 17 produce ampicillin-resistant colonies. These cloned fragments are further characterized by restriction enzyme size determination and mapping and their ability to cross-hybridize with the labeled fragment of S. cerevisiae HIS4 (hybrid13

EN 205 627 Β Post wash washing with 2xSSC at 55 ° C) a). Example I, Chapter G. Each HIS4-containing plasmid contains one or more fragments that hybridize to the S. cerevisiae HIS4 gene. Such an HTS4-containing plasmid is promoted to give the HIS4-containing plasmid pYJ8, shown in Figure 25. Plasmid pYJ8 contains pBR325 sequences including the chloramphenicol and ampicillin resistance genes and the Pichia HIS4 gene.

The ARG4 gene was isolated from P. pastoris NRRL Y-1130 by the same procedure and S2072 from S. cerevisiae strain Arg [arg 4, leu 2, trp 1, gal 2 mutant; strain available from Yeast Genetic Stock Center (Berkeley, CA)].

One skilled in the art will recognize that other marker genes from Pichia can be similarly isolated using appropriately deficient strains of S. cerevisiae.

Isolation of autonomic replication sequences of Pichia pastoris.

Other useful components of the vectors of the present invention are Pichia-derived autonomous replication sequences (PARS), which increase both the transformation frequency of GS115 (NRRL Y-15851) and the sustainability of the plasmid as a stable extrachromosomal element.

To search for Pichia ARSs, DNA from Pichia pastoris GS115 (NRRL Y-15851) was partially digested with Taql and fragments between 5 and 10 kbp were isolated and cloned into the unique ClaI site of pYJ8 Δ Cla (see Figure 26). figure). Plasmid DNA was recovered from about 10,000 His ⁺ Pichiat colonies and used to transform E. coli. Plasmids were isolated from approximately 10,000 ampicillin-resistant colonies and transformed back into GS115. From this "subdirectory" transformation, 40 His ⁺ yeast colonies were separately yeast-selective and grown in separate cultures in selective media. Total yeast DNA was extracted from each of these 40 cultures and transformed into E. coli. Two plasmids, pYA63 (PARS1) and pYA90 (PARS2), whose yeast DNA preparations produce cultures of E. coli most resistant to ampicillin, were selected for further analysis. Both plasmids transform Pichia pastoris GS115 (NRLL Y-15,851) at very high frequencies and each contains foreign DNA insertions.

To the degree of ability of ARSs to maintain the plasmid as an autonomous element in Pichia, cultures of yeast cells transformed with one of the plasmids are grown in selective medium and occasionally sampled. The status of plasmid sequences in cells is determined by Southem hybridization of unrestricted yeast DNA to radiolabeled pBR325. Plasmids pYA63 and pYA90 persist in Pichia for at least 10 generations (but integrate up to 50 generations) in selective media.

One of the putative Pichia autonomous replication sequences (PARS 1) was cloned into several other Pichia vectors to test its ability to retain transforming DNA as an autonomous element. Plasmids pYJ30 (Figure 27) and pBPJ1 (Figure 34) are still present as autonomous elements on selective media (His') after 20 generations of growth and are present in multiple copies per cell. Southem blot analysis of cells transformed with pYJ30 indicates approximately 10 copies per cell.

Plasmid pSAOH5 (Figure 18) and pT76H4 (Figure 22b), which contain PARS 1 provided by pYJ30 and pBPJ1, are determined to have similar stability to the plasmids they are derived from. Strains containing these vectors were selectively grown (His) for about 50 generations in the presence of glucose. The cells are then transferred to non-selective conditions (His ⁺ ) and the loss of prototrophy is observed. The stability of these plasmids is comparable to that of pYJ30, including the rapid loss of His-prototrophy upon transfer to a non-selective medium. Thus, it is believed that experiments with plasmids containing the autonomous replication sequence PARS 1 result in gene expression from autonomous plasmid DNA.

Constructs containing a new $ galactosidase gene.

In order to demonstrate the ability of the regulatory regions of the present invention to regulate the production of protein products, a novel DNA construct is constructed. Thus, the E. coli lacZ gene is transferred to a number of plasmids, under the control of the regulatory regions of the gene encoding the p72 (alcohol oxidase) or p76 polypeptide. The construction of plasmids pSAOH1, pSAOH5, pSAOH10, pTAFH.85, pT76H1, pT76H2, pT76H3 and pI76H4 is described in XIV. example.

Although the introduction of the regulatory region-β-galactosidase gene of the present invention into a host yeast cell with a plasmid as a carrier, it will be understood by those skilled in the art that it is not necessary to introduce the regulatory region structural construct into the cell by plasmid. Thus, the regulatory region structural gene constructs of the present invention can also be manipulated by vectors other than plasmids. Alternatively, the regulatory region structural gene construct may be integrated into the yeast chromosome of the host.

It will be apparent to those skilled in the art that the scope of the present invention is not limited to the production of β-galactosidase under the control of the regulatory domains described herein. The various polypeptides that may be produced under the control of the regulatory domains of the invention are limited only by the imagination of the reader. There are several methods for constructing DNA sequences that encode the desired polypeptides, e.g. oligonucleotides of different lengths can be synthesized by known methods. Many of these oligonucleotides can be fused to their specific bases14

EN 205 627 Β due to its pairing properties into longer, double-stranded molecules. The oligonucleotides that make up the double-stranded molecule can be joined (ligated) with a DNA ligase enzyme. Alternatively, a DNA molecule having the desired coding sequence may be synthesized using a reverse transcriptase enzyme, using the messenger RNA for the desired polypeptide as a template for the reverse transcriptase action. Another possibility is to clone genomic DNA fragments and monitor whether the desired product is expressed.

The DNA sequence encoding the desired polypeptide can be modified by a variety of methods to construct a regulatory region structural gene construct. so e.g. the ends of the DNA prepared as described above can be ligated to short double-stranded DNA molecules containing a nucleotide sequence recognized by specific restriction endonucleases by DNA ligase, a so-called "linker" molecule. After ligation, digestion of these molecules produces ends corresponding to a specific restriction endonuclease recognition site at the ends of the prepared DNA sequence.

The β-galactosidase gene construct of the three specific regulatory regions prepared in the course of this work is described using the restriction map shown in Figures 15 and 16. The restriction map depicted in Figure 15a depicts a construct consisting of a 0.85 kilobase pair HindIII / BamHI portion of the pPG 6.0 5 'regulatory region and an E. coli lacZ gene (the 3.6 kilobase pair BamHI-Nru fragment shown). . The same construct is present in all plasmids pTAFH.85, pT76H1 and pT76H2, which are described in more detail below (see Example XIV). The restriction map depicted in Figure 15b describes a construct consisting of a 1.3 kilobase pair HindIII / EcoRI portion from the pPG 6.05 'regulatory region and the lacZ gene from E. coli. The same construct is present in all plasmids pT76U1, pT76H4, which are described in more detail below (see Example XIV).

A16. Fig. 3A is a restriction map of a construct consisting of a 1.1 kilobase EcoRI-BamHI fragment from the 5 'regulatory region of pPG 4.0 and the lacZ gene from E. coli. The same construct is present in plasmids pSAOH1, pSAOH5 and pSAOH10, which are described in more detail below (see Example XIV).

Plasmid pSAOH1 is schematically illustrated in Figure 17. In addition to the regulatory region containing the fusion product of the β-galactosidase gene detailed in Figure 16, plasmids have been shown to contain:

(a) pBR322 sequences including the Amp ^R gene;

(b) the Pichia pastoris HIS4 gene;

(c) 2 μ circular DNA of S. cerecisiae;

(d) the interrupted URA3 gene from S. cerevisiae.

The plasmid therefore has the ability to be transformed and replicated in E. coli and yeast hosts. Selectable markers for DNA manipulation are present in all E.

coli, both in the yeast host.

Plasmid pSAOH5 is schematically illustrated in Figure 18. The plasmid is similar to the pSAOH1 described above, except that some of the S. cerevisiae circular DNA and DNA flanking the Pichia pastoris HIS4 gene are missing while an autonomously replicating sequence of Pichia pastoris (PARSl) is added. apYA63 from).

Plasmid pSAOHIO is schematically illustrated in Figure 19. The plasmid contains:

(a) a regulatory region of a β-galactosidase gene fusion product;

(b) genes transmitting pBR325 sequences, including tetracycline resistance, chloramphenicol resistance, and ampicillin resistance (tet ^R and cam ^R and amp ^R , respectively); and (c) the S. cerevisiae HIS4 gene (obtained from pYA2 as described below).

The plasmids pTAFH.85, pT76H1 and pT76H2 are analogous to the three plasmids described above, except that the regulatory region used is the β-galactosidase fusion product described in Figure 15a (instead of the fusion product described in Figure 16).

Plasmids pT76H3 and pT76H4 are analogous to plasmids pSAOH1 and pSAOH5, except that the regulatory region used would be a fusion of the β-galactosidase gene to the product of Figure 15b (instead of the fusion product of Figure 16).

Plasmid pTAFH.85 is schematically illustrated in Figure 20 and includes:

(a) the regulatory region of Fig. 15 a) fusion product of β-galactosidase gene;

(b) pBR322 sequences including the amp ^R gene;

(c) the Pichia pastoris HIS 4 gene;

(d) S. cerevisiae 2 μ circular DNA; and (e) an interrupted URA3 gene from S. cerevisiae.

Plasmid pT76H1 is schematically illustrated in Figure 21 and includes:

(a) the regulatory region shown in Figure 15a for the β-galactosidase gene fusion product;

(b) pBR322 sequences including the amp ^R gene;

(c) the Pichia pastoris HIS4 gene and the autonomously replicating sequence (PARS1).

Plasmid pT76H2 is schematically illustrated and included in Figure 22;

(a) the fusion product of the β-galactosidase gene of the regulatory region shown in Figure 15a;

(b) genes transmitting pBR325 sequences including tetracycline resistance, chloramphenicol resistance, and ampicillin resistance; and (c) the S. cerevisiae HIS4 gene.

Plasmid pT76H3 is schematically illustrated in Figure 22a and includes:

(a) the regulatory region shown in Figure 15b for the β-galactosidase gene fusion product;

(b) pBR322 sequences including the amp ^R gene;

(c) the Pichia pastoris HIS4 gene;

(d) S. cerevisiae 2μ circular DNA, and

(E) the interrupted URA3 gene from S. cerevisiae.

Plasmid pI76H4 is schematically illustrated in Figure 22b and includes:

(a) a fusion product of the β-galactosidase gene of the regulatory region shown in Figure 15b;

(b) pBR322 sequences including the amp ^R gene;

(c) the Pichia pastoris HIS4 gene; and (d) the Pichia pastoris autonomous replication sequence (PARS1).

Expression of $ galactosidase in yeast.

Pichia pastoris GS115 (NRRL Y-15,851) was transformed with the novel β-galactosidase gene containing construct described above. Numerous transformed yeast strains were deposited with the United States of America Department of Agriculture, Northern Region Research Center under the following deposit numbers:

host plasmid The deposit number of the transformed strain GS115 pSAOH 1 NRRL Y-15 852 GS115 pSAOH 5 NRRL Y-15 853 GS115 pSAOHIO NRRL Y-15 854 GS115 pTAFH.85 NRRL Y-15 855 GS115 pT76H 1 NRRL Y-15 856 GS115 pT76H 2 NRRL Y-15 857

The novel β-galactosidase gene-containing constructs can also be used to transform E. coli. Atransformed bacterial strains were also deposited with the Northern Region at the Research Center Institute (Peoria, Illinois) under the following numbers:

host plasmid The deposit number of the transformed strain MC1061 pSAOH 1 NRLLB-15 861 MC1061 pSAOH 5 NRRLB-15,862 MC1061 pSAOHIO NRRL B-15 863 MC1061 pTAFH.85 NRRL B-1564 MC1061 pT76H 1 NRRL B-15,865 MC1061 pI76H 2 NRRLB-15 866 MC1061 pTAO 13 NRRL B-15 875 MC1061 pI76H 3 NRRLB-15,800 MC1061 pI76H 4 NRRLB-18 001 MC1061 pI76U 1 NRRLB-18 002

The first eight plasmids containing the alcohol oxidase and the p76 regulatory region of the invention containing the lacZ gene fusion product were transformed and grown to a stationary phase in Pichia pastoris GS115 strain (NRRL Y-15,851) in minimal medium supplemented with biotin and glucose as a carbon source. When the cells reach the stationary phase, they are transferred to a minimal medium supplemented with biotin and methanol as a carbon source. After the cells grow for about 3-5 generations at 30 ° C, they are transferred to fresh minimal medium supplemented with biotin and grown on glucose or methanol as the sole carbon source. At defined times, samples are withdrawn from the cultures and analyzed for the presence of β-galactosidase and alcohol oxidase in VH. and XV. by methods detailed in the Examples.

It has been found that cells grown on glucose as the sole carbon source do not produce detectable levels of β-galactosidase or alcohol oxidase, whereas cells growing on methanol as the sole carbon source express significantly both alcohol oxidase and β-galactosidase. In addition, glucose-grown cells are found to express measurable amounts of alcohol oxidase and β-galactosidase when exposed to carbon sources. Thus, it is clear that the regulatory regions of the present invention are susceptible to both the presence of methanol and the conditions for carbon starch.

To demonstrate that the regulatory region of the invention is susceptible to growth on non-catabolite repressing carbon source and to carbon source starvation conditions, an alcohol oxidase regulatory domain plasmid (pTA013) and a p76 regulatory domain plasmid (pI76U1) are used in a non-methanol utilizing yeast strain. One of the transformed strains used, designated SEY2102-pTA013, was deposited with the Northern Region at the Research Center Institute (Peoria, Illinois) under the accession number NRRL Y-15858. Saccharomyces cerevisíae NRRL Y-15858 and SEY2102-pT76H1 were grown on glucose, ffuctose, ethanol, glycerol and galactose for about five generations and then exposed to carbon source conditions. Standard measurement of β-galactosidase (see Example XV) after five generations indicates that glycerol and galactose-grown cells produce large amounts of β-galactosidase, whereas glucose and ffuctose-grown cells essentially do not produce β-galactosidase. When β-galactosidase is measured 6 hours after carbon source starvation, the production of moderate amounts of β-galactosidase by transformed organisms grown on glucose and ffuctose is observed, while significant amounts of β-galactosidase produced by glycerol and galactose-grown cells are observed. Thus, the regulatory domains of the invention are capable of regulating the production of protein products in genetically diverse yeast hosts and are not limited to adaptation to methanol utilizing strains.

EXAMPLES

The composition of the following buffers and solutions is given below (the percentages given are by weight):

Mole Tris Buffer: 121.1 g Tris base in 800 mL water; adjusting the pH to the desired value by adding aqueous hydrochloric acid; allowing the solution to cool to room temperature before making the final pH adjustment and adjusting the solution to a final volume of 1 liter.

HU 205 627 Β

S buffer: 1.5 M sorbitol in 0.04 M sodium phosphate buffer, pH 6.6.

PK buffer: 0.14 M NaCl,

1% sodium dodecyl sulfate (SDS),

0.01 M EDTA

0.05 M Tris buffer pH 8.4.

ETS buffer: 10 mM EDTA,

0.2% SDS

0.01 M Tris buffer, pH 7.6.

TE buffer: 1.0 mM EDTA

0.01 mol / L in Tris buffer, pH 7.4.

SSC: 0.15 mol / L NaCl, mmol / L sodium citrate adjusted to pH 7.0 with NaOH.

TAE: 40 mM acetic acid, EDTA EDM

0.02 M Tris buffer pH 8.3.

PBS (phosphate buffered saline): 10 mM sodium phosphate, pH 7.0,

0.15 M NaCl.

Laemmli loading buffer: 62.5 mmol / L Tris-HCl, pH 6.8,

2% SDS,

10% glycerol,

5% 2-mercaptoethanol,

0.01% bromophenol blue.

RIPA buffer: 1% NP40 (S igma),

1% sodium deoxycholate,

0.1% SDS in PBS.

X SSPE: 20 mM EDTA,

0.16 M NaOH,

0.2 M NaH 2 PO 4 .H 2 O,

3.6 M NaCl adjusted to pH 7.0 with NaOH.

Denhardt's solution (50 x): 5 g Ficoll, g polyvinylpyrrolidone, g bovine serum albumin (BSA; Pentax V. Fraction)

Make up to 500 ml with water.

Pre-Hybridization Buffer: 5 X SSPE, x Denhardt's solution,

50% deionized formamide,

0.2% SDS,

200 μg / ml of goat and denatured herring sperm DNA.

LB (Luria-Bertani) medium: 5 g Bacto tripton, g Bacto yeast extract,

2.5 g NaCl per liter of water, adjusted to pH 7.5 with NaOH.

YPD medium: 1% Bacto yeast extract,

2% Becto peptone, 2% glucose.

SD medium: 6.7 g of "Yeast Nitrogen Base without Amino Acids" (DIFCO),

2% glucose per liter of water.

Sediment: 1 mol / liter sorbitol, mmol / liter EDTA, mmol / liter DTT,

SCE buffer: 9.1 g sorbitol,

1.47 g of sodium citrate,

0.168 g EDTA, ml water was adjusted to pH 5.8 with HCl.

CaS: 1 mol / l sorbitol, sterilized with a Ca / 2 filter.

PEG solution: 20% polyethylene glycol-3350, sterilized by filtration with CaCl2, mmol / L Tris-HCl, pH 7.4.

SOS: 1 mol / liter sorbitol,

0.3 x YPD medium, mmol / L CaCl2.

Formamide dye mixture: 0.1% "xylene cyanol FF",

0.2% bromophenol blue, mmol / liter EDTA,

95% deionized formamide.

Cover gel: 76.8 g urea, ml acrylamide stock, 8mll0xTBE

Make up to a final volume of 160 ml.

Acrylamide starting material: 38 g of acrylamide, supplemented with g of bis (N, N-methylene-bis-acrylamide) water to a total volume of 100 ml.

Bottom gel: 14.4 g urea,

3.0 g sucrose,

7.5 ml 10xTBE,

4.5 ml of acrylamide feedstock,

0.3 ml bromophenol blue solution (0.01 g / ml) made up to 30 ml with water.

Pre-Hybridization Buffer for Hybridization Selection: 50% formamide,

0.75 mol / liter NaCl,

0.1 M Tris pH 7.4,

0.008 mol / liter EDTA,

HU 205 627 Β

0.5% SDS,

200 g / ml rabbit liver tRNA (Sigma).

Oőmól / literesNETSpuffer. 0.5 M NaCl, Molar EDTA, Tris pH 7.4,

0.2% SDS.

IOxRTpuffer: 500 mM NaCl,

340 mmol / L Tris, pH 8.3, mmol / L MgCl ₂ , mmol / L DTT (dithiothreitol).

dilRT: 4glH ₂ O, μΐ10xRT buffer, μΐ reverse transcriptase, 15 units g / gl (Life Sciences, Ins.).

dideoxy:

ddATP: 0.49 mmol / L, dd CTP: 0.1165 mmol / L, dd GTP: 0.369 mmol / L, dd TTP: 0.82 mmol / L, dNTP Mix: 0.625 mmol / L dGTP,

0.625 mmol / liter dATP,

0.625 mmol / liter ΊΤΡ.

Chase: 1.125 mmol / liter dATP,

1.125 mmol / liter dCTP,

1.125 mmol / liter dGTP,

1.125 mmol / L in IT'P, 1x RT buffer.

Unless otherwise indicated, the above concentrations represent the base (1x) concentration used. In the examples where different concentration levels are used, this fact is denoted by referring to the multiplication of the base (1x) concentration.

In the examples, the following abbreviations are used:

EDTA ethylenediaminetetraacetic teraecetsav TEMED Ν, Ν, Ν ', N' -tetramethylene diamine DTT DTT BSA bovine serum albumin EtBr ethidium bromide ci Curie dATP deoxyadenosine triphosphate dGTP deoxyguanosine triphosphate TTP thymidine triphosphate dCTP deoxycytidine triphosphate ICAM cDNA from HL "Generic" deoxy triphosphate nucleotide Oligo (dT) 12-18

Source: Collaborative Research, Inc.

Zymolyase 60,000

Source: Miles Laboratories

Enzymes are used under the conditions recommended by the manufacturers.

Many procedures are performed on a routine basis, following standard descriptions, which will be detailed herein.

The centrifugation is carried out for such a time and at a speed that a clear supernatant is obtained. Generally, the yeast cells are centrifuged at at least 1500 g for at least 5 minutes.

The nucleic acids are extracted with an equal volume of a 50: 48: 2 by volume mixture of phenol: chloroform: isoamyl alcohol and an equal volume by volume of a 48: 2 mixture of chloroform: isoamyl alcohol.

When using gels, filters, etc. it is described as washing or soaking in a special solution, immersing the entire gel, strainer, or the like in a suitable container (tray, cup, tube, etc.), so that the gel, strainer, etc. contacting the entire surface with said solution.

Ethanol precipitation of the nucleic acids is accomplished by first adjusting the salinity of the solution containing the nucleic acid and then contacting the solution with two volumes of cold ethanol.

Example I

Growth and recovery of yeast cells

Pichia pastoris NRRL Y-1130 was grown in a carbon source under limited conditions in a continuous culture at 30 ° C, i.e., IMI salt containing methanol or ethanol as the sole carbon source in minimal medium as described by Wegner in U.S. Patent 4,414,329. . The minimum IMI nutrient weight per 1 liter of medium is 36 mmol KH ₂ PO ₄ , 23 mmol (NH ₄ ) ₂ SO ₄ , 2 mmol MgSO ₄ , 6.7 mmol KCl, 0.7 mmol CaCl _2. 0.2 gm of CuSO ₄ .5H ₂ O, 1.25 gm of Kl, 4.5 gm of MnSO ₄ , 2 gm of Na ₂ MoO ₄ , 0.75 gm of H ₃ BO ₃ 17.5 gmol contains ZnSO ₄ by weight, 44.5 gmol cent FeCl ₂ and 1.6 gmol biotin. Cells growing on methanol were grown to a cell density of 140 g / l (dry weight) for approximately 12 hours. Cells growing on ethanol were grown to a cell density of 90 g / l with a holding time of about 11.5 hours. When methanol or ethanol is added to the fermentor, a stock stock solution containing 20% and 45% alcohol is used.

g of fermented Pichia pastoris cells were collected by centrifugation and resuspended to about 10 ⁸ cells / ml in 0.1 M Tris pH 8.0 containing 1% 2-mercaptoethanol. These cells were incubated for 5-10 minutes at 37 ° C and harvested by centrifugation. The pellet was washed once with 30 mL of S buffer and resuspended in 5 mL of S buffer per cell. Zymolase (Miles Biochemicals) was added to the cell suspension to reach a final concentration of 500 µg / ml. The cells were incubated at 37 ° C and centrifuged; discarding the supernatant and collecting the cell pellet. This pellet was frozen in liquid nitrogen and stored at -70 ° C for later use.

II. example

Isolation of yeast RNA

Whole cell RNA was prepared by pulverizing the frozen pellet described in Example 1 in a mortar with a pestle and then crushing the frozen pellet for 2 to 5 minutes in a Waring Blender mixer with liquid nitrogen.

EN 205 627 Β. The powdered pellet was added to PK buffer

At a cell concentration of 7.5 ml / g. Proteinase (Boehringer Mannheim) was added to the resuspended pellet to a final concentration of 400 pg / ml and the suspension was incubated at room temperature for 10 minutes. This mixture was extracted with phenol / chloroform / isoamyl alcohol followed by chloroform / isoamyl alcohol. The nucleic acids are precipitated by addition of NaCl up to 0.25 mol / L and addition of ethanol. The pellet is resuspended in a minimal volume of ETS buffer, i.e., a volume sufficient to dissolve the nucleic acids; this usually means 100 pg-1 mg DNA per ml solution. This solution was re-extracted with phenol / chloroform / isoamyl alcohol and then precipitated with chloroform / isoamyl alcohol and finally ethanol.

The nucleic acids are redissolved in a minimum volume of TE buffer. RNA in this solution was enriched either by centrifugation on 4 mL of CsCl "cushion" [1 g CsCl / mL, 1 mM EDTA, 10 mM Tris (pH 7.4) buffer] or by precipitation such that the solution is made up to 2 M per liter of LiCl, kept at 4-8 ° C overnight, and collected by centrifugation. Poly A ⁺ RNA was recovered from the solution by affinity chromatography on an oligo (dT) cellulose column. Generally, 0.25 g of oligo (dT) cellulose (type 3, Collaborative Research) is prepared for chromatography based on 5-10 mg total RNA. 0.25 g of oligo (dT) cellulose is slurried with 2 ml of ETS buffer and filled into a small, siliconized glass column. This oligo (dT) cellulose column is washed by layering 10 ml of a 0.1 M solution in oligo (dT) cellulose and allowing the wash solution to flow through the oligo (dT) cellulose matrix. The oligo (dT) cellulose was washed in the same way with 10 mL of ETS buffer and last washed with 10 mL of 0.5 M NETS buffer.

Total RNA (5-10 mg) was resuspended in ETS buffer at a concentration of up to 10 mg / ml in a water bath at 65 ° C for 2 minutes and immediately placed on ice. The RNA solution was then allowed to warm to room temperature and 5 M NaCl stock solution was added to give a final salt concentration of 0.5 M NaCl in the RNA solution. The resulting RNA solution was layered on the prepared oligo (dT) cellulose column and allowed to flow slowly through the column at a rate of about 1 drop / 5 seconds. The spillage at the bottom of the column was collected in a tube and layered again on top of the column. The material collected from the bottom of the column was layered on top again a second time, yielding an RNA solution that had a total of three passes through the oligo (dT) cellulose column. After the final passage, the material is collected and labeled polyA-RNA, i.e., non-polyA-RNA. The column is then washed with 30 mL of 0.5 M NETS and finally the polyA ⁺ RNA is eluted from the column by loading 5 mL of ETS buffer and allowing this buffer to slowly flow through, collecting the polyA + ⁺ RNA fraction in a 5 ml fraction from the bottom of the column. Assuming no NaCl in the polyA ⁺ RNA fraction, the NaCl concentration of the fraction is adjusted to 0.25 mol / L and the RNA is precipitated with ethanol.

III. example

Creating a cDNA library

Complementary DNA (cDNA) clones are synthesized as follows. Poly A ⁺ RNA prepared as described in Example H. In 10 g resuspended μΐ 7 H ₂ O and then adjusted to a final concentration of 2.7 mmol / L CH ₃ HgOH, then incubated at RT for 5 minutes. The first cDNA strand was synthesized at 42 ° C for 15 minutes in 50 μΐ solution of 50 mM Trist (pH 8.3 at 42 ° C), 10 mM MgCl ₂ , 30 mM 2 mercaptoethanol, 70 mM KCl, 500500 gm / d dATP, dGTP and TTP, 200 pmol / liter dCTP, 25 pg / ml oligo (dT), 60 µg / liter ml contains 25 units of actinomycin Dt, 25 units of RN-azine (Biotec, Inc,), 25 gCi of α- ³² P-dCTP (32.5 pmol) and 120 units of reverse transcriptase (Life Sciences, Inc.). The reaction mixture was incubated at 37 ° C for an additional 15 minutes. The reaction was stopped by the addition of 2 μΐ 0.5 M EDTA and 0.5 μΐ 20% SDS. The reaction mixture was adjusted to 0.3 M NaOH and incubated at 65 ° C for 30 minutes. The reaction mixture was then neutralized by the addition of 10 μΐ 1 M Tris pH 7.4 and the reaction mixture was adjusted to a concentration of 0.21 M HCl. The reaction mixture was then extracted with phenol / chloroform / isoamyl alcohol then chloroform / isoamyl alcohol and finally collected on a Sephadex G50 column in TE buffer and concentrated to 100 μΐ by either butanol extraction or centrifugation under vacuum. The single-stranded cDNA was precipitated from the concentrated solution with ethanol, the cDNA was collected by centrifugation, and resuspended in 100 μΐ water.

The aqueous single-stranded cDNA solution was adjusted to a concentration of 2.5 M ammonium acetate, precipitated with ethanol, collected by centrifugation and resuspended in 20 μΐ water. This single stranded DNA solution was brought to a final volume of 50 μΐ 50 mmol / l potassium phosphate buffer containing 5 mmol / l MgCl ₂ -t, 1 mmol / l 2-mercaptoethanol, 250 to 250 pmol / L of each dATP, dGTP and TTP, 125 g / l dCTP, 25 pCi-a- ³² P-dCTP (32.5 pmol) and 8 units of DNA Poll Klenow fragment (New England Biolabs). The resulting reaction mixture was incubated at 37 ° C for one hour in order to synthesize the complementary second strand of DNA for the single-stranded cDNA. The reaction was quenched by the addition of 2 μΐ 0.5 M EDTA. The double-stranded cDNA was extracted with phenol / chloroform / isoamyl alcohol then extracted with chloroform / isoamyl alcohol and chromatographed on a Sephadex G50 column in TE buffer. The double-stranded cDNA fractions were collected and the collected material was concentrated and precipitated as described for single-stranded DNA.

After the final ethanol precipitation and centrifugation of the double-stranded cDNA,

Resuspend in water and adjust to a final volume of 50 μΐ with 50 mM Iris (pH 8.3 at 42 ° C) containing 10 mM MgCl ₂ , 30 mM 2-mercapto- ethanol, 70 mM KCl, 500 M dXTP and 150 units reverse transcriptase. The resulting solution was incubated at 42 ° C for 15 minutes to ensure the completion of the second strand cDNA synthesis. The reaction was stopped and concentrated by the addition of 2 μΐ 0.5 M EDTA and precipitated as described for the single-stranded cDNA reaction.

The double stranded cDNA pellet was resuspended in 42 μΐ H ₂ O and the solution brought to a final volume of 47 μΐ in 5 μΐ stock solution to which 2.8 mol / l NaCl 200 mmol / L of NaOAc and 45 mmol / of ZuSO ₄ and the pH is adjusted to 4.5 at 22 ° C. To separate the hairpin loop, three separate reactions were performed with three different concentrations of Si nuclease (Sigma). 1 unit, 10 units or 100 units of S1 nuclease was added to the reaction mixture to make 50 μΐ and the reaction was incubated at 22 ° C for 30 minutes. The reaction was 2 μl 0.5 M EDTA and 2.67 μΐ 2 mol / liter of Tris base. 6 μg of rabbit liver tRNA was added as a promoter and the reaction mixture was concentrated and precipitated as described previously, except that the DNA pellet was incubated in ΤΕ-buffer instead of water. After the final precipitation, the pellet was resuspended in 20 μΐ TE buffer and made up to 50 μΐ final concentration with teminal transferase buffer (BRL) containing 10 pmoles α- ³² P-dCTP, 2 pmol / L dCTP and 21 units. contains terminal transferase (Ratliff Biochem). The resulting solution was incubated at 37 ° C for 30 minutes to attach a poly (d / C) tail to the 3 'end of the double stranded cDNA. The reaction was quenched with 5 μ 5 0.5 M EDTA, extracted, chromatographed and stored as an ethanol precipitate.

The double-stranded d / C-tailed cDNA was either re-bound to pBR322, open at the PstI site, or first size-fractionated on a Sepharose CL 4B-200 column (25 μΐ fractions). For the non-fractionated "library", 150 ng of double-stranded cDNA with poly (d / C) tails was bound in 180 μΐ 10 mM Tris pH 7.4 - 0.1 M NaCl and EDTA- ra for 1 mM 900 ng d / G-tailed pBR322 with a PstI site. To each 25-ml fraction of the fractionated "library" was added 125 ng of pBR322 with poly (d / G) tails in a final volume of 50 μΐ of the same composition as described above. The binding reaction mixture was incubated at 65 ° C for 3 minutes and then at 42 ° C for 2 hours and then allowed to cool slowly to room temperature.

The bound cDNA "library" was bound to a suitable E. coli LE392 (ATCC 33 572) as follows: LE392 inoculum was grown overnight at 37 ° C in 2 x LB medium. 5 ml of this overnight culture were inoculated into 200 ml of fresh 2 x LB medium and grown at 37 ° C until the OD 4 0 was 0.2-0.3. This culture was placed in ice for 10 minutes and the cells were harvested by centrifugation at 4 ° C. The cell pellet was resuspended in 80 ml of ice-cold 0.1 M CaCl ₂ and incubated for 25 minutes at 4 ° C. Cells were harvested by centrifugation at 4 ° C, resuspended in 2 ml of ice-cold 0.1 M CaCl ₂ and incubated for at least 2 hours at 4 ° C before use. Next, 200 μΐ of the appropriate cell is used for 50 μΐ of the “binding mixture” for transformation. The appropriate cells and DNA are pooled and ca. Incubate at 4 ° C for 10 minutes, then incubate at 37 ° C for 5 minutes, and finally place the mixture in ice for 10 minutes. An equal volume of 2 × LB medium was added to the transformation mixture and incubated at 37 ° C for 45 minutes. Transformed cells are plated at 250 μΐ / plate on 150 μm 2 x LB plates containing 15 pg / ml tetracycline. The plates were then incubated at 37 ° C for 24 hours and stored at 4 ° C.

We make replica disks by "sealing" the nittocellulose filters on an original filter to remove the colonies from the plate. These replica plates are incubated on 2X LB-Tet plates (15 pg / ml tetracycline). The colonies on the filter were prepared for screening by transferring the filters to 2 x LB-Tet plates containing 200 pg / ml chloramphenicol, incubating the filters at 37 ° C for at least 12 hours and then lysing the colonies. for rinsing the filters in an aqueous collecting vessel for 10 minutes, the rinse water contained 1.5 M NaCl and 0.5 M NaOH. The filters were then neutralized by rinsing in an aqueous collector flask for 15 minutes, containing 1.5 M NaCl and 0.5 M Tris pH 7.4; this neutralization rinse is repeated once more. The filters were then air dried and finally vacuum dried for 2 hours at 70 ° C.

ARC. example

Colony hybridization

Vacuum-dried nittocellulose filters containing the cDNA library (prepared as in the previous examples) were pre-hybridized at 42 ° C for 5 hours in pre-hybridization buffer. The filters were removed from the pre-hybridization buffer and gently rubbed with gloved hands into 5 x SSPE to remove cell debris. Filters were placed in hybridization buffer (identical to pre-hybridization buffer except 1x Denhardt's solution) or ³² P-labeled single-stranded cDNA (10 ⁶ rpm / ml) digested with S1 nuclease under milder conditions. polyA ⁺ mRNA isolated from Pichia pastoris grown in methanol or ethanol was hybridized to the filters for 17 hours at 42 ° C. After hybridization, the filters are briefly washed in 2 x SSPE at 22 ° C,

EN 205 627 Β followed by two washes at 65 ° C in 0.1 x SSPE, both for 10 minutes,

PolyA ⁺ mRNA end labeling was performed by adding 2 µg of polyA ⁺ mRNA to 50 μΐ of 50 mM iris (pH 9.5), heating at 100 ° C for 3 minutes, and rapidly cooling on ice. This RNA solution was diluted to a final volume of 200 μΐ and adjusted to 50 mM Tris pH 9.5, 10 mM MgCl2, 5 mM DTT, and 50 pmole ³² P-cc-ATP. Ten units of T4 polynucleotide kinase (Boehringer Mannheim) were added and the mixture was incubated at 37 ° C for 1 hour. The kinase reaction was quenched with 10 μΐ 0.5 M EDTA, extracted with phenol / chloroform / isoamyl alcohol, and chromatographed on Sephadex G50 to remove any unlabeled radioactive label.

Example V

Northern hybridization

2-5 µg of polyA ⁺ mRNA was heated at 65 ° C for 5 minutes in 10 mM sodium phosphate buffer (pH 7.4) containing 50% formamld, 2.2 mol / L formaldehyde and 0.5 mM / liter of EDTA. The resulting solution was cooled to room temperature and 5 x "sample buffer" (0.5% SDS, 0.025% bromophenol blue, 25% glycerol, 25 mmol / l) was added (usually 0.2 volume relative to the volume of treated sample). liter EDTA). Samples were applied to a 1.5% agarose gel in 10 mM sodium phosphate buffer (pH 7.4) containing 1.1 M formaldehyde and electrophoresed in the same buffer. The gel was acridine orange (33 g / ml), stained with 10 mM sodium phosphate buffer (pH 7.4), the gel was rinsed for 10 minutes in the same buffer, and the RNA was transferred to nitrocellulose by rinsing in 10 x SSPE for at least 10 minutes. VI. Example.

VI. example

Isolation of genomic DNA and clans

Pichia genomic DNA was isolated as shown in Figure II. using the method described for isolating Pichia RNA. The nucleic acid pellet was resuspended in a minimal volume of TE buffer and incubated with 20 µg / ml RNase A for 30 minutes at 37 ° C. The solution was adjusted to a concentration of 0.14 M NaCl and treated with proteinase K at 200 µg / ml for 15 minutes at 22 ° C. The resulting solution is extracted first with phenol / chloroform / isoamyl alcohol and then with chloroform / isoamyl alcohol and finally precipitated with ethanol. The precipitated DNA was resuspended in minimal volume of TE buffer and centrifuged to purify the DNA solution.

gg of Pichia genomic DNA prepared as described in the previous paragraph was digested with various restriction enzymes (BRL) and electrophoresed on a 1% agarose gel containing TAE. The DNA fragments are denatured on the gel by soaking the gel in 1.5 M NaCl and 0.5 M NaOH for 20 minutes. The gel is soaked in 10X SSPE for at least 5 minutes prior to transfer. A nitrocellulose plate is cut to size of the gel, moistened with water and soaked briefly in 10 x SSPE. This filter is placed on top of the gel, which in turn has been placed on a piece of parafilm. A Whatman filter paper sheet and a paper towel stack are placed on top of the nitrocellulose to extract the DNA from the gel and transfer to the nitrocellulose. A weight is placed on the bundle to facilitate transfer. The DNA is allowed to migrate in this manner for at least 3 hours. After transfer, the filter was briefly rinsed with 5 x SSPE, air dried, and vacuum dried at 70 ° C for 2 hours. Complementary genomic fragments were identified by hybridization to cDNA clones subjected to nick translation of pPC 8.0, pPC 6.4 and pPC 15.0, using the same pre-hybridization, hybridization, and wash buffers as described in Figure IV. example.

200 ng of the cDNA clone was subjected to nick translation at 14 ° C in 30 μΐ of 50 mM Tris-HCl pH 7.4, 10 mM MgSO ₄ , 100 pmM DTT. 50 pg / ml BSA, 2020 pmoles / liter dGTP, TTP and dATP, 31.25 pmoles ³² Pa-dCTP (3200 Ci / mmol, NEN), 2 units E. coli It contains DNA Poll (BRL) and 0.02 ng DNase I. The reaction was quenched by addition of 1 μΐ 0.5 M EDTA and 1 μΐ 20% SDS. The labeled DNA solution was adjusted to 0.3 M NaOH and placed in hot water for one minute. This mixture was chromatographed on a Sephadex G50 column. The labeled DNA fraction is collected, the specific activity is determined and the assay sample is used in hybridization experiments.

The genomic fragments that hybridize to the cDNA probe samples were isolated by digesting 2,000 pg of the Pichia genomic DNA with the previously used restriction enzymes (BRL) and electrophoresing on a 1% agarose gel in TAEpuffer. Lanes corresponding to fragments hybridizing with cDNA clones were excised from the agarose gel, DNA was electroeluted, passed through an Elutip column (Schleicher and Schull) and precipitated with ethanol.

The electroeluted fragments were resuspended in water and 200 ng of the fragment was ligated to 500 ng of pBR322, which was cleaved at appropriate restriction sites and dephosphorylated if necessary. The ligation reaction was performed in 300 µl of 66 mM Tris pH 7.4, 6.6 mM MgCl ₂ , 10 mM DTT, 0.4 mM ATP, μg / ml BSA and 4080 units T ₄ DNA ligase and incubated at ° C for 24 hours. The ligation mixture is directly transformed into appropriate E. coli LE392 cells. The cells are adapted for reaction and transformed as described in Example III. 3 series of transformations were performed with 10, 40 and 100 ng of pBR322 (plus insert), using 100 μΐ of appropriate cells for each transformation. Cells are plated as in IH. except that the antibiotic selection was carried out with 50 pg / ml ampicillin. The clones are transferred

EN 205 627 Β nitrocellulose, replicate and prepare for hybridization IH. The filters are screened for the appropriate nick translated cDNA fragments. Plate-purified colonies that are positive for hybridization are used to construct additional plasmids as follows.

Plasmid-bearing E. coli LE392 is grown to 1.0 OD 50 in 1x LB medium containing 50 pg / ml ampicillin and grown overnight with the addition of chloramphenicol to a final concentration of 200 pg / ml. Cells were washed with 0.8% NaCl, 20 mM Trist, pH 8.0, 20 mM EDTA, and lysozyme treated with 25% sucrose, 50 mM Trist, pH 7.4 ) In a solution containing 20 mM EDTA and 450 pg / ml lysozyme. Lysis is achieved by the addition of 1 M NaCl to a final concentration of 2.0 M, followed by the addition of an equal volume of 0.2% Triton X-100 and 40 mM EDTA. The preparation was purified by rotation at 20,000 rpm for 45 minutes. The supernatant is then extracted with a phenol / chloroform / isoamyl alcohol mixture, then extracted with chloroform / isoamyl alcohol and precipitated with ethanol. The pellet was resuspended in TE buffer, treated with RNase A, extracted with phenol / chlorophone / isoamyl alcohol and then extracted with chloroform / isoamyl alcohol. Solid CsCI was added to give a final concentration of 800 pg / ml and EtBr was added to a final concentration of I mg / ml. The resulting solution was spun in a Vti 50 rotor at 49,000 rpm / min for 18-20 hours at 20 C, ^e.

Plasmid strips are visualized by UV fluorescence and recovered from the tube with a needle and injection syringe. The plasmid solution was extracted four times with n-butanol and precipitated with ethanol at -20 ° C. The ethanol precipitation was repeated at least twice more to remove all CsCl. The plasmid was stored at -20 ° C as ethanol precipitate.

VII. example

Purification of alcohol oxidase

Protein samples obtained from Pichia pastoris cells grown on methanol as described in Example I are prepared by lysing yeast cells followed by purification centrifugation to remove cellular debris as follows. A portion of the fermetor fluid was removed and adjusted to pH 7.5 with ammonium hydroxide and homogenized on a Dyno-Mill Model KDL using a 0.6 liter vessel at 30 ° C in continuous operation using a 3 # strap combination and hour flow. The beads in the mill are lead-free glass beads with a diameter of 0.3-05 mm. The resulting homogenate was centrifuged at 5 ° C and 20,000 g for 30 minutes to obtain a cell-free supernatant.

Six 130 ml fractions of the cell-free supernatant were placed in a cellulose acetate dialysis bag and dialyzed against 5 liters of distilled water at 5 ° C. After 4 days, the aqueous phases of each bag are decanted. The solids remaining in the bags contain two types of solids. The thin white top layer is carefully removed and discarded. The lower solid is brownish-yellow in color and is a crystalline alcohol oxidase. A portion of the crystalline alcohol oxidase is redissolved in distilled water (approximately ten times the volume of the solid) and the dye peroxidase assay shows 94 enzyme units / ml. The specific activity of the alcohol oxidase is 10.4 enzyme units / mg protein.

The crystalline precipitate formed from the dialysis described above is dialyzed against 0.05 M potassium phosphate pH 7.5 and applied to a 2x200 cm column equilibrated in Sephacryl 200 (Pharmacia). 3.5 ml fractions were collected at a flow rate of 10 ml / h and the alcohol oxidase activity was measured.

The alcohol oxidase activity for the reaction with methanol is determined by the following assay (dye peroxidase method). A dye-buffer mixture was prepared by mixing 0.1 ml o-dianizidine solution (1 wt% o-dianizidine in water) with 12 ml aeration of 0.1 M sodium phosphate buffer, pH 7.5. The assay mixture is prepared from 2.5 ml dye-buffer mixture, 50 µl methanol, 10 µ 10 peroxidase solution [1 mg horseradish peroxidase (Sigma, type H)] and 25 µΐ alcohol oxidase solution. The assay mixture is maintained at 25 ° C in a 4 x 1 x 1 cm cuvette and the dye-induced increase in absorbance at 460 nm is recorded for 2 to 4 minutes. The enzyme activity is calculated using the following formula:

Activity (pmoles / min / ml or enzyme units / ml) ~ -min x 11.5 where 11.5 is a factor based on a standard curve made with known aliquots of H ₂ O ₂ and ΔΑ is the change absorbance during the experimental interval.

Alcohol oxidase content of 0.1 µg of total protein from each fraction was measured by gel electrophoresis on SDS-polyacrylamide (12%).

VIII. example

DNA and protein fraction analysis

DNA sequencing is performed by the dideoxy chain extension method using M13 bacteriality (Sanger et al., 1980) or by chemical modification (Maxam-Gilbert, 1980). DNA fragments corresponding to the 5 'end of the alcohol oxidase gene are inserted into the vectors M13mp8 and M13mp9, or end-labeled for chemical modification using restriction enzyme sites available in the art.

The 710 bp HindIII / SalI fragment from pPG 4.0 was terminated for Maxam-Gilbert sequence analysis by first digesting 33 µg of plasmid with Hind III. The reaction mixture was extracted with a phenol / chloroform / isoamyl alcohol mixture and then with chloroform-iso

It is extracted with a mixture of amyl alcohol and is precipitated with ethanol. DNA was collected by centrifugation and resuspended in 31 μΐ water. 100 μθί ³² P-ct-dCTP (3200 Ci / mmol) and two units of Poll Klenow fragment were added to the reaction mixture to give a final volume of 50 μΐ and the mixture was adjusted to finally 400 μΐ / liter of dATP , 400 μπιόΐ / liter dGTP, 50 mM Trist (pH 7.4), 10 mM MgSO ₄ and 1 mM DTT. The reaction mixture was induced at 37 ° C for 1 h and quenched by the addition of 2 μ ED 0.5 M EDTA. The mixture is then extracted with phenol / chloroform / isoamyl alcohol, then extracted with chloroform / isoamyl alcohol, then chromatographed on a Sephadex G-50 column with chloroform / isoamyl alcohol, and the labeled nucleic acid fractions are collected and precipitated with ethanol. After centrifugation, the DNA pellet was resuspended in water and digested with SalI. The digested material was subjected to electrophoresis on a 1% agarose gel in TAE buffer, and the 710 bp band was excised, the DNA was electroeluted, extracted with butanol and precipitated with ethanol. The fragment was resuspended in 100 μΐ TE buffer, adjusted to 2.5 M ammonium acetate, and precipitated with ethanol. The resulting DNA fragment was resuspended in TE buffer at 50,000 cpm / μΐ.

The four base-altering reactions are carried out as follows:

a) Incubate the G (guanine) reaction mixture for 8 minutes at 22 ° C with 1 μΐ (50,000 cpm) of the labeled DNA fragment, 4 μΐ of water, 200 ml of 50 mM sodium cacodylate, pH 8.0. Contains 1 mM EDTA (DMS buffer) and 1 μΐ dimethyl sulfate. The reaction was stopped by adding 50 ml of DMS-stop buffer containing 1.5 M sodium acetate (pH 7.0), 1 M 2-mercaptoethanol and 100 μg / ml tRNA, followed by ethanol. (750 μΐ) was added to the reaction mixture and the reaction mixture was kept at -70 ° C for at least 15 minutes.

b) Incubate the G / A (guanine / adenine) reaction mixture for 10 minutes at 22 ° C containing 2 μΐ (100,000 cpm) of the labeled DNA fragment, 8 μΐ of water, and 30 μΐ of formic acid. The reaction was stopped by addition of 200 μΐ Hz stop buffer [0.3 M sodium acetate (pH 5.5), 0.1 M EDTA and 25 μg / ml tRNA] followed by ethanol (750 μΐ). and the reaction mixture is kept at -70 ° C for at least 15 minutes.

c) The T / C (thymidine / cytosine) reaction mixture was incubated for 10 minutes at 22 ° C containing 2 μί (100,000 Cpm) of the labeled DNA fragment, 18 μΐ of water and 30 μg of hydrazine. The reaction is stopped in the same manner as described in (b) above.

d) Incubate the C (cytosine) reaction mixture for 10 minutes at 22 ° C containing 1 μΐ (50,000 cpm) of the labeled DNA fragment, 4 μΐ of water, 15 μg of 5 M NaCl and 30 μΐ of hydrazine. The reaction is stopped as described in b).

The DNA pellet was collected by centrifugation, resuspended in 250 μΐ 0.3 M sodium acetate (pH 5.5), and ethanol precipitated with 750 μΐ 95% ethanol. The pellet was collected by centrifugation, dried in vacuo for 5 minutes, and the DNA was cleaved by resuspending the pellet in 100 μΐ 1: 10 (v / v) piperidine. The cleavage reaction mixture was incubated at 90 ° C for 30 minutes and stopped by adding 500 μΐ of 98% ethanol, 60 mM sodium acetate (pH 5.5) and 10 μg / ml tRNA. / ethanol bath (about -70 ° C) for about 5 minutes and the DNA fragments collected by centrifugation. The fragments were resuspended in 50 μΐ 0.3 M sodium acetate (pH 5.5) and ethanol precipitated with 100 μΐ 95% ethanol. This ethanol precipitation was repeated, the pellet was washed with 95% ethanol, centrifuged and concentrated in vacuo. The pellet was resuspended in a solution of 10 μΐ of 80% formamide, 10 mM NaOH, 1 mM EDTA, 0.1% xylene cyanol and 0.1% bromophenol blue. 2-3 μΐ was subjected to electrophoresis on a 10% 0.4 mm thick polyacrylamide gel in TBE buffer.

The amino acid sequence of the alcohol oxidase was determined by Sequemat, Inc. (Watertown, Massachussets) using 2 mg of purified alcohol oxidase from Pichia pastoris. The first 18 amino acids of the mature protein show the following sequence (without the initial Met): Ala-Ile-Pro-Glu-Glu-Phe-Asp-Ile-LeuVal-Leu-Gly-Gly-Gly-Ser-Gly-Ser.

IX. example

Determine the origin of alcohol oxidase transcription

To determine where the start point of the alcohol oxidase mRNA is located, a primer extension experiment was performed using a synthetic oligonucleotide copied from the 5 'end DNA sequences of the alcohol oxidase gene as a primer and 10 μg of polyA ⁺ Pichia pastoris mRNA as a template. 10 μg of Pichia pastoris polyA ⁺ mRNA was combined with 3 ng of primer (5'-CTT CTC AAG TTG TCG-3 ') in a final volume of 9.3 μΐ containing 43 mM NaCl, 29.2 mM Trist. (pH 8.3), contains 5.2 mM MgCl ₂ and 4.3 mM DTT. The nucleic acids are denatured at 70 ° C for 5 minutes and then re-pooled with slow cooling to 22 ° C. The reconstituted mixture was placed in a tube containing 4 μΐ dNTP mix, 0.8 μί RT buffer and 1 μί ³² P-ct-dCTP (800 Ci / mmol). 3 μΐ of this mixture is added to 1-1 μΐ of each appropriate dNTP. Add the last 3 μΐ of the mixture to 1 μΐ of water. Reactions were initiated by addition of 1 μΐ dilRT and induced at 42 ° C for 15 minutes. The reaction mixture was treated with 3 μΐ Chase RT at 42 ° C for 15 minutes. Reactions were terminated by the addition of 7.5 μΐ formamide-dye mixture and 45 μ elektr subjected to electrophoresis on a 0.4 mm thick gradient gel in 1 x TBE. After electrophoresis, the gel was fixed in 10% acetic acid with 10% methanol at 20 rpm

HU 205 627 Β cent. The gel was dried under vacuum and used to expose the XAR X-ray film.

The gradient gel is prepared as follows: 300 μΐ 10% ammonium persulfate and 14 μΐ TEMED are added to 30 mL of overlay gel; Add 75 μΐ of 10% ammonium persulfate to 7 mL of bottom gel, remove 6 mL of top gel with a pipette, and then remove 6 mL of bottom gel with the same pipette. The gel is poured around the gel plates, followed by 22 ml of top gel.

Example X mRNA hybridization selection and in vitro translations

Positive hybridization-translation experiments were performed by linearizing 20 pg of cloned Pichia genomic DNA (prepared as described in Example VI) with various restriction endonucleases. This DNA was denatured by adjusting the solution to 0.3 M NaOH and incubating at 65 ° C for 10 minutes. The denatured DNA solution was rapidly cooled on ice and neutralized to 0.5 M Tris-HCl (pH 7.4). An equal volume of 20 x SSPE is added to the denatured DNA immediately before the DNA is bound to the nitrocellulose filter. Prior to transfer to the nitrocellulose filters (Schleicher and Schuell BA83.9 mm diameter), the filters are prepared with H _2. Moistened with O, boiled for 10 minutes, then rinsed three times in 10 x SSPE. 10 µg of DNA were then bound to each filter by transferring the DNA to the filter and air drying under vacuum at 70 ° C for 2 hours.

Prior to pre-hybridization, the filters were placed with bound DNA in 1 ml sterile water and heated for 1 minute at 100 ° C, cooled on ice, rinsed with 1 ml sterile water in a Vortex and 1 ml pre-hybridization buffer, then 40 pg (2 pg / ml ETS). ) polyA ⁺ mRNA is added directly to the pre-hybridization buffer. The hybridization mixture was heated at 65 ° C for 10 minutes and then incubated at 42 ° C for 24 hours.

After hybridization, the filters were washed twice briefly in 1 x SSPE containing 0.5% SDS at 22 ° C, then seven times in 1 x SSPE containing 0.5% SDS at 50 ° C. at 5 ° C for 5 minutes per rinse, then three times in 0.1 x SSPE at 50 ° C for 5 minutes, and once in 0.1 x SSPE at 65 ° C for 10 minutes. The RNA was eluted from the filter by boiling for 1 min with 300 µl H ₂ O containing 15 µg of rabbit liver tRNA. The eluted RNA was rapidly frozen in a dry ice-ethanol bath. The RNA mixture was allowed to warm to room temperature and the filters were removed. The RNA mixture was then precipitated by adjusting the medium to 2.5 M ammonium acetate and precipitating twice with ethanol and finally resuspending the precipitate in 100 µl of water before lyophilizing.

Translations of the above RNA are performed using standard techniques known to those of ordinary skill in the art, e.g. the guidance provided in the guidance provided with the in vitro rabbit reticulocyte lysate translation kit provided by New England Nuclear. The protein products were subjected to electrophoresis on an 8% polyacrylamide gel containing 4.5% bundled gel.

XI. example

Antiserum preparations and immunoprecipitation

An antiserum to rabbits containing both p72 (alcohol oxidase) and p76 polypeptide derived from Pichia pastoris cells was prepared using standard procedures as follows: The extracts were dialyzed against PBS before injection. In an 8-week procedure, each rabbit receives 3 injections, each containing 1 mg of total protein, in a volume of 0.1 ml along with 0.2 ml of complete Freund's adjuvant. The injections are given subcutaneously at 6-10 sites in the rabbit. At the end of the eighth week, the rabbits are bled and these sera are assayed for the presence of purified Pichia pastoris alcohol oxidase antibody by the Ouchterlony double diffusion procedure, whereby the antigen and antibody are diffused with one another and form an immune complex on the agar. (Basic and Clinical Immunology, 1987, Ed: D. P. Stites, J. D. Stobo, J. V. Wells.)

Purified rabbit anti-p72 (alcohol oxidase) and anti-p76 protein antibodies are prepared by affinity chromatography of whole antiserum over CN72r-linked p72 (alcohol oxidase) p76 Sepharose 4B column (Pharmacia). 1 g of CNBr-activated gel is made to hydrate the gel for 15 minutes in 200 ml of mmol / h HCl, followed by washing in 3 x 50 ml of switching buffer [0.1 mol / L sodium carbonate (pH 8) and 0.5 mol / l / liter NaCl]. 5 ml of p72-p76 6 mg / ml solution (in switching buffer) was added to the gel and stirred gently overnight at 4 ° C. Unbound protein was removed by rinsing with 3x50 mL of coupling buffer. The remaining active groups were removed by incubation with 1 M ethanolamine (pH 8) for 2 hours. The non-covalently bound material is removed from the gel by washing with 50 mL of 2 mM sodium thiocyanate (in PBS). The gel was finally washed with PBS (3 x 50 mL) prior to antiserum chromatography. 5 ml of purified anti-p72-p76 antiserum was mixed with the gel and incubated with gentle agitation for 2 hours at 4 ° C. The antiserum-gel mixture was then pipetted onto a 1 x 8 cm column and washed with 15 mL of PBS. The purified antibody was eluted from the column with 6 mL of 2M sodium thiocyanate (in PBS). After elution from its branches, the purified antibody is vigorously dialyzed against PBS containing 0.02% sodium azide.

The antisera purified by affinity chromatography were added to an in vitro translation mixture in PBS (supplemented with 1% NP40) and incubated overnight at 4 ° C. The antigen-antibody complex is precipitated with Pansorbin (Calbiochem) on ice for 2.5 hours. Pansorbin is prepared by washing in RIPA buffer. There are four Pansorbin precipitates24

Wash with RIPA buffer and Laemmli loading buffer before electrophoresis.

XII. example

Transformation of Pichia pastoris

A) Cell growth.

1. A colony of Pichia pastoris GS115 (NRRL Y-15 851) is inoculated for approx. 10 ml of YPD medium and the culture is shaken at 30 ° C for 12-20 hours.

2. After about 12 to 20 hours, the cells are exposed to ca. Dilute 0.01-0.1 OD ₆₀₀ and keep cells in hanging growth phase in YPD medium at 30 ° C for approx. 6-8 hours.

3. After about 6-8 hours, 100 ml of YPD medium is inoculated with 0.5 ml of inoculum culture for approx. 0.1 OD 4 (or equivalent). The culture was shaken for 12-20 hours at 30 ° C.

4. When the culture reaches an OD ₆₀₀ of about 0.2-0.3 (after about 16-20 hours), the culture is recovered by centrifugation at 1500 g for 5 minutes.

B) Preparation of spheroplasts

1. Wash cells once with 10 ml sterile water. (In steps 1-5, each centrifugation is performed at 1500 g for 5 minutes).

2. Wash cells once with 10 ml freshly prepared SED.

3. Wash cells twice with 10 ml sterile 1 M sorbitol.

4. The cells are resuspended in 10 ml of SCE buffer.

5. Add 5-10 µl of 4 mg / ml Zymolyase 60,000 (Miles Laboratories). Cells were incubated at 30 ° C for 30-60 minutes.

Since spheroplast preparation is a critical step in the transformation process, spheroplast preparation should be monitored as follows. Aliquots of 100 μΐ cells were added to 900 μl of 5% SDS and 900 μΐ of 1 M sorbitol solution before or immediately after addition to the zymolase and at various times during the incubation period. The incubation is stopped at the point where cells lyse in SDS but not lysate in sorbitol (usually between 30 and 60 minutes of incubation).

6. Wash spheroplasts twice with sterile 1 M sorbitol solution by centrifugation at 1000 g for 5-10 minutes. (The time and speed of centrifugation may vary; the centrifugation must be sufficient to allow the spheroplasts to enter the sediment, but not so much as to be crushed by force).

7. Wash cells once in 10 ml sterile CaS.

8. The cells are resuspended in a total of 0.6 ml CaS.

C) transformation

1. Transfer the transforming plasmid DNA samples (up to 20 μΐ volume) into 12x75 mm sterile polypropylene tubes. (DNA in water or TE buffer; it is recommended to add approximately 1 μΐ of 5 mg / ml sonicated E. coli DNA to each sample for maximum transformation frequency).

2. Add 100 μΐ spheroplast to each DNA sample and incubate at room temperature for approx. For 20 minutes.

Add 3.1 ml of PEG solution to each DNA sample and incubate at room temperature for approx. For 15 minutes.

4. The samples are centrifuged at 1000 g for 5-10 minutes and the PEG solution is decanted.

5. The samples are resuspended in 150 μΐ SDS and incubated for 30 minutes at room temperature.

6. Add 850 μΐ sterile 1 M sorbitol solution and place aliquots of the samples on a plate as described below.

D) Regeneration of spheroplasts

1. Specification for Preparation of Regenerating Agar Medium.

(a) Agar sorbitol: 9 g Bacto agar, 54.6 g sorbitol and

Water (240 g) was autoclaved.

b) 10 x glucose: 20 g of glucose and 100 ml of water are autoclaved.

c) 10 X SSC: 6.75 g of yeast nitrogen base (without amino acids) and 100 ml of water are autoclaved (any necessary amino acid or nucleic acid can be added up to 200 μg / ml before or after autoclaving).

d) 30 ml of 10 x glucose and 30 ml of 10 ΧΔ SSC are added to the molten agar sorbitol solution. 0.6 ml of 0.2 mg / ml biotin solution and up to 20 mg / ml are added any desired amino acid or nucleic acid. The Regenerating Agar is kept in a molten state at 55-60 ° C.

2. Plate the transformation samples on a plate. A bottom layer of agar was discarded from 10 ml of regeneration agar per plate at least 30 minutes before the transformation samples were ready. Aliquots of 10 ml of regeneration agar are dispensed into tubes in a 45-50 ° C bath while the transformation samples are in SDS. Aliquots of the transformation samples described above are added to tubes filled with regeneration agar and poured onto the bottom agar layer of the plates by transferring an appropriate volume of sample into 10 ml molten regeneration agar at 45-50 ° C and regenerating 10 ml solid bottom agar layer. of agar.

3. Determination of the quality of the spheroplast preparation.

Take 10 μΐ of a sample and dilute 100-fold with 990 μΐ of 1 mol / l sorbitol solution. Take 10 μΐ of the 100-fold dilution and further dilute 100-fold by adding a second 990 μΐ of 1 mol / l sorbitol solution. 100 μΐ of each dilution was plated onto YPD agar plates to determine the concentration of whole cells remaining in the preparation which did not become spheroplast. 100 μΐ of each dilution is added to 10 ml regeneration agar (supplemented with 40 μg / ml histidine) to determine all regenerable spheroplasts. Transformation experiments yielded good results: 1-3 x 10 ⁷ total regenerable spheroplasts / ml and 1 x 10 ³ whole cells / ml.

HU 205 627 Β

4. Incubate the plates at 30 ° C for 3-5 days.

XIII. example

Isolation of Pichia pastoris HIS4 gene and autonomous replication sequences The strains

The strains used are:

(a) Pichia pastoris strain NRRL Y-1130;

b) Pichia pastoris GS115 (his4; NRRL Y-15,851);

c) S. cerevisiae strain 5799-4D (his4-260 his4-39; NRRL Y-15,859); and

d) E. coli strain 848 (F 'met thi gal T, ^R 080 ^s hsdR hsdM ⁺ ).

B) Plasmids pYA2 (see Figure 23, composed of a 9.3 kilobase PstI fragment containing the S. cerevisiae HIS4 gene inserted at the PstI site of pBR325) is the source of the S. cerevisiae HIS4 gene fragment deposited in an E. coli host. and is available to the public under the accession number NRRL B-15 874.

YEpl3 is available from the American Type Culture Collection under accession number ATCC 37,115.

C) Media

Pichia pastoris is grown on YPD (rich) or IMG (minimal) medium. IMG, which is a minimal medium, consists of:

1. IM _L salts: at final concentration of 36.7 mmol / L KH ₂ PO ₄ , 22.7 mmol / L (NH ₄ ) ₂ SO ₄ , 2.0 mmol / L MgSO ₄ .7H ₂ 0.6,7 mmol / liter KC1,0,7 mmol / l CaCl ₂ 2H ₂ O, prepared as a ten-fold concentrated stock solution and autoclaved;

2. Trace Salts at a final concentration of 0.2 gmol / L CuSO ₄ .5H ₂ 0,1,25 gmol / L KI, 4.5 gmol / l MnSO ₄ .h O _2, 2.0 pmol / L Nam ₄ .2H ₂ O, 0.75 pmol / l H3BO3,17,5 pmol / L ZnSO ₄ .7H ₂ O, 44.5 pmol / l FeCl ₃ .6H ₂ O, prepared as a 400x stock solution and filter sterilized;

3. 0.4 pg / ml biotin; and

4. 2% glucose.

E. coli is cultured in either LB medium or 2B medium (0.2% NH ₄ PO ₄ , 1.2% Na ₂ HPO ₄ , 0.013% MgSO ₄ .7H ₂ O, 0.074% CaCl _2. 2H ₂ O, pg / ml thiamine and 0.4% glucose) supplemented with 100 pg / ml tryptophan and 0.2% casaminic acid (hydrolyzed casein).

D) Isolation of ADNS

I. Large-scale production of yeast DNA.

DNA preparation of both Pichia pastoris and S. cerevisiae was performed by growing yeast cells in 100 ml of minimal medium until Aqq 1-2 was achieved and the cells were harvested by centrifugation at 2000 g for 5 minutes. The cells were washed once in water, once in SED, once in 1 M sorbitol, and resuspended in 5 mL of 0.1 M TrisHCl, pH 7.0, in 1 M sorbitol. Cells were mixed with 50-100 µl of 4 mg / ml Zymolyase 60,000 (Miles Laboratories) and incubated at 30 ° C for 1 hour to analyze the cell walls. The spheroplast composition was then centrifuged at 1000 g for 5-10 minutes and resuspended in "lysis buffer" (0.1% SDS, 10 mM Tris-HCl, pH 7.4), 5 mM EDTA and 50 mM per liter of NaCl). Proteinase K (Boehringer Mannheim) and RNase A (Sigma) were added at 100-100 µg / ml and the mixture was incubated at 37 ° C for 30 minutes. The DNA was deprotected by gentle mixing with an equal volume of chloroform containing isoamyl alcohol (24: 1 v / v), and the phases were separated by centrifugation at 12,000 g for 20 minutes. The upper (aqueous) phase is aspirated into a fresh tube and extracted with an equal volume of phenol / chloroform / isoamyl alcohol. The phases are separated as above and the upper phase is placed in a 2-3 volume cold tube containing 95% ethanol. The sample is mixed gently and the DNA is collected by wrapping it on a plastic rod. The DNA was immediately dissolved in 1 mL of TE buffer and dialyzed overnight at 4 ° C against 100 volumes of TE buffer.

2. Small-scale production of yeast DNA.

ml of yeast culture in minimal medium was grown until the Α ^ θθ value was 1-5 and the cells were harvested by centrifugation at 2000 g for 5 minutes. Cells were resuspended in 1 ml SED and transferred to a 1.5 ml microcentrifuge tube, washed once with 1 M sorbitol solution, and resuspended in 0.5 ml 0.1 M Tris-HCl, pH 7.4 ), to which sorbitol was added 1 M Zymolyase 60,000 (10 (from 4 mg / ml solution)) and the cells were incubated for 3060 minutes at 30 [deg.] C. The cells were then centrifuged for 1 minute in "lysis buffer". resuspended and incubated at 60-70 C. After 15 minutes, the samples were mixed with 100 µl of 5 M potassium acetate, kept in an ice bath for 15 minutes and centrifuged for 5 minutes. The supernatants were decanted into a fresh microcentrifuge tube containing 1 ml of 95% ethanol, mixed and centrifuged for 10 seconds. Finally, the DNA pellet was air dried for 10-15 minutes and then dissolved in 50 µl of TE buffer.

3. Large-scale isolation of E. coli DNA.

For large scale (0.5 to 1 liter) plasmid preparationE. cultures were grown at 37 ° C in shaking medium 2B supplemented with the above antibiotic and the appropriate antibiotic. For cells containing plasmids derived from pBR322, the cultures are grown to about 0.7 A ₅₅₀ , then sufficient chloramphenicol is added to give a concentration of 100 µg / ml and the cells are harvested after about 15 hours. Strains containing plasmids derived from pBR325 are inoculated into supplemented 2B medium for about 10 minutes. Incubate at 0.01-0.05 at A ₅₅₀ and shake at 37 ° C for 20-24 hours before harvest.

4. Small-scale production of E. coli DNA

For small-scale rapid plasmid isolation, 2 ml cultures were grown in supplemented 2B medium with antibiotic, and overnight shaking followed by centrifugation yielded 1.5 ml cells.

EN 205 627 Β in microcentrifuge tubes. Plasmids from the preparations were isolated according to the alkaline lysis method described by Bimboim and Doly (1979 Nucl. Acids. Res. 7, 1519-1523).

E) Isolation of restriction DNA fragment

Restriction enzymes were obtained from New England Biolabs and Bethesda Research Laboratories and digested as directed by the supplier. Restriction mapping is performed by comparing parallel-digested products of plasmids with inserted DNA and without inserted DNA. The restriction fragments were purified from the agarose gel by electroelution onto Whatman 2MM strips of paper. The fragments were recovered from the paper by 3-4 washes with 0.1-0.2 ml of 0.1M NaCl, 50 mM Tris-HCl pH 8.0 and 1 mM Finally, the fragments are extracted with phenol / chloroform / isoamyl alcohol, precipitated with ethanol and redissolved in a small amount of TE buffer.

F) Creation of a P. pastoris Library "in E. coli.

For the construction of the Pichia pastoris DNA-YEpl3 "library", 100 µg of YEpl3 was completely digested with BamHI and treated with calf intestinal alkaline phosphatase to remove the terminal 5 'phosphate from the DNA. A 100 pg aliquot of wild-type Pichia pastoris DNA from Pichia pastoris NRRL Y-1130 was partially digested with 10 units of Sau3AI for 5 minutes at 37 ° C in a total volume of 1 ml. Fragments between 5 and 10 kilobases are sized by centrifugation by centrifugation at 5-20% sucrose. One µg of the vector and 2 µg of the Pichia Sau3AI fragment were mixed with 20 units of T4 DNA ligase (Bethesda Research Laboratories) in a total volume of 200 µl and incubated overnight at 4 ° C. The ligated DNAs were transformed into E. coli by adding the complete ligation reaction mixture to 2 ml of the appropriate E. coli 848 cell culture and incubating at 0 ° C for 15 minutes. The mixture was heated at 37 ° C for 5 minutes, then 40 ml of LB medium was added and incubation continued at 37 ° C for an additional 1 hour. Ampicillin was then added at a total concentration of 100 pg / ml and incubated for an additional 1 hour. Finally, the cells are centrifuged at 3000 g for 10 minutes, resuspended in 1 ml of fresh LB medium and plated in equal aliquots on 10 LB agar plates containing 10 pg / ml ampicillin. The approximately 50,000 colonies which resulted were scraped from the plates and a portion of the cells was inoculated with 500 ml of the supplemented 2B medium, about ₅₅₀ 0,1Α starting value. The culture was grown and the plasmid was extracted as described above. Of the colonies collected from the library, 96 of the 100 colonies tested contained tetracycline-sensitive and 7 of the 10 colonies tested contained plasmids with inserted DNA.

For the construction of the Pichia pastoris DNA - pYJ8 ΔCla library, 50 pg pYj8Ca was completely digested with ClaI and treated with calf intestinal alkaline phosphatase to remove terminal 5 'phosphate from the DNA.

A 100 pg aliquot of DNA from Pichia pastoris NRRL Y-15 851 was partially digested with 20 units of Taql for 5 minutes at 65 ° C in a total volume of 1 ml. Fragments between 5 and 10 kilobase pairs are sized by electroelution from a 0.5% agarose gel (see Example II, Chapter E). One pg of the vector and 2 pg of the Pichia pastoris Taq1 fragment were mixed with 20 units of T4 DNA ligase (Bethesda Research Laboratories) in a total volume of 200 µl and incubated overnight at 4 ° C. The ligated DNAs were transformed into E. coli by adding the complete ligation reaction mixture to 2 ml of the appropriate E. coli 848 cell culture and incubating for 15 minutes at 0 ° C. The mixture is heated at 37 ° C for 5 minutes, then 40 ml of LB medium is added and incubation is continued for 1 hour at 37 ° C. Ampicillin was then added at a concentration of 100 pg / ml and incubated for an additional hour. Finally, the cells are centrifuged at 3000 g for 10 minutes, resuspended in 1 ml of fresh LB medium and plated in equal aliquots on 10 LB agar plates containing 100 pg / ml ampicillin. Approximately 10,000 colonies that are formed are scraped from the plates and a portion of the cells are inoculated into 500 ml of supplemented 2B medium for about 1 hour. 0.1 at baseline A ₅₅₀ . The cultures were grown and the plasmid was extracted as described above.

G) Southern hybridization

To transfer large or highly coiled DNA molecules to nitrocellulose, the DNA is first partially hydrolyzed by soaking the agarose gel with 0.25 M HCl before denaturing with alkali. Hybridization of labeled fragments from the S. cerevisiae HIS4 gene to Pichis pastoris DNA was performed in the presence of 50% formamide, 6 x SSC, 5 x Denhardt's solution, 0.1% SDS, 1 mM EDTA and 100 pg / ml denatured herring sperm DNA. finally at 42 ° C. Post-hybridization washes were performed in 1 x SSC, 1 mM EDTA, 0.1% SDS and 1.0% sodium pyrophosphate at 65 ° C. The DNA is labeled with ³² P in the same way as for alV. example.

H) DNA sequence analysis

DNA sequence analysis was performed by the dideoxy nucleotide chain termination method of Sanger et al. (1980) (J. Mol. Biol. 143, 161-178).

I) Transformation of yeast

Transformation of S. cerevisiae is performed by the spheroplast-forming method of Hinnen et al., 1979 (Proc. Nat. Acad. Sci. USA 75,1929-1933).

Transformation of Pichia pastoris is performed following the procedure described above.

J) Analysis of Pichia pastoris transformants

The ability of each plasmid to survive as an autonomous element in Pichia pastoris cells was determined by picking a transformant colony from the regeneration agar plate and extending it onto SD IM medium and inoculating it into liquid IMG medium. The SD plates were incubated at 30 ° C and then plated on a single colony

Dissolve from this plate, spread on a second SD plate and inoculate a second flask with 1MG medium. This process is repeated a third time. The three IMG cultures were grown by shaking at 30 ° C until the Agoo value was ca. 1-2 cells and the cells are harvested by centrifugation. DNA from yeast cultures was extracted as described above, subjected to electrophoresis at 30 volts and 30 milliamps for 10 to 15 hours on a 0.8% agarose gel, transferred to nitrocellulose and hybridized to ³² P-labeled pBR322 or pBR325, this is described above. As a control, a sample containing 10 ng of plasmid isolated from E. coli and a sample containing 1-2 pg of untransformed GS115 DNA were subjected to electrophoresis in parallel with the experimental sample.

K) Isolation of Pichia DNA sequences.

DNA fragments containing the Pichia HIS4 gene are isolated from a Pichia DNA "library" for their ability to be complementary to S. cerevisiae his4 strains. The library is YEpl3 S. cerevisiae-E. coli "commuter" vector consists of partially digested 5-20 kb Sau3 AI fragments of wild-type Pichia DNA inserted into BamHI. Spheroplasts of S. cerevisiae NRRL Y-15,859 (strain 5799-4D; his4ABO) were prepared by the method of Hinnen et al. (1978), mixed with the Pichia DNA library and allowed to regenerate in a histidine-deficient medium. Transformation results in approximately 1 x 10 ³ prototrophic yeast colonies out of 5 x 10 ⁷ total regenerable spheroplast populations. Total yeast DNA was extracted from 20 His * colonies and transformed into E. coli. Of the yeast DNA preparations, 17 produce ampicillin-resistant colonies and each contains a plasmid consisting of YEpl3 + inserted DNA. To confirm that the His * transforming gene contains the Pichia HIS4 gene and a non-suppressor activity DNA fragment, the restriction digestion products of the plasmids were hybridized to the labeled DNA fragment containing a large portion of the S. cerevisiae HIS4 gene and washed lightly. Each of the plasmids complementing his4 S. cerevisiae strain contains sequences that hybridize to the S. cerevisiae HIS4 gene.

To search for DNA fragments containing Pichia ARS activity, DNA from Pichia pastoris GS115 (NRRL Y-15,851) was partially digested with Taq1 and fragments between 5 and 10 kilobase pairs were isolated and pYJ8 Δ Cla. clal (see Figure 26). Plasmid DNA was recovered from approximately 10,000 His * 4 Pichia colonies and used to transform E. coli. Plasmids were isolated from approximately 10,000 ampicillin-resistant colonies and re-transformed into GS115. The 40 His * yeast colonies from this sub-library transformation were transferred to separate selective media and grown in separate cultures in selective media. Total yeast DNA from each of these 40 cultures was extracted and transformed into E. coli. Two plasmids, pYA63 (PARS1) and pYA90 (PARS2), whose yeast DNA preparations produce the most ampicillin-resistant colonies of E. coli, are selected for further analysis. Each of these plasmids transforms Pichia pastoris GS115 (NRRL Y-15).

851) at very high frequencies and each one contains a foreign DNA insert.

XIV. example

Regulatory Area Creation of a lacZ gene fusion product

A) p72 (alcohol oxidase) regulatory region constructs. Plasmid pPG 4.0, a vector pBR322 containing the 4 kb EcoRI-PvuII genomic DNA fragment from Pichia pastoris, was cleaved with Pstl, treated with S1 nuclease to form "blunt" ends where the cleavage occurs and then BamHI- cleavage to obtain a 1.12 kbp DNA fragment containing the alcohol oxidase regulatory region and coding information for the first 15 amino acids of the alcohol oxidase. This 1.12 kbp DNA fragment has the following nucleotide sequence:

B

1.12 kbp, with "S1 nuclease" .... CTA GGT GGT G 'treated end ............... GAT CCA CCA CCT AG |

Alcohol Oxidase B Leu _n GIy ₁₂ Glyi ₃

This 1.12 kbp was ligated into pSEY101 E. coli-S. cerevisiae "shuttle" vector to EcoRI / Smal / BamHI (cleaved with BamHI and Smal) [Douglas et al. (1984) Proc. Nat. Acad. Sci. U.S. 81, 3983-3987]. The vector ApSEY101 contains the E. coli lacZ gene and a "poly-linker" having the following nucleotide sequence:

Rí Sm B

II

R _t Sm B Val ₉ Val ₁₀ pgalactosidase The above ligation yields the pTAOll hybrid plasmid (see Figure 29).

The lacZ gene fusion product of the pTAOll regulatory region is out of phase with respect to β-galactosidase production, as can be seen from a comparison of the E sequence with the two sequences:

This is the sequence

R, B | 1.12 kbp j

... GAATTCCG ..... CTA GGT GGT GGA TCC CGT GGTT ...

CrTAAGGG GATCCACCACCrAGGGCAGCAA ... ..... ...

f i

R, B

Leu,, Gly _f2 Gly, ₃ Gly ₁₄ Ser ₁₅ Arg Arg The plasmid pTAOll is cleaved at its unique BamHI site and the following Smal linker inserted:

HU 205 627 Β

Sm j

GATCACCCGGGT

TGGGCCCACTAG i

Sm thus creates a hybrid vector pTA012 having the nucleotide sequence of the regulatory region for the lacZ fusion product:

F sequence

R [Sm

1.12 kbp ί

GAATTCCC ............. CTA GGT GGT GGA TCA CCC GGG TGA TCC CGT CGT

CITAAGGG ........... GAT CCA CCA CCT AGT GGG CCC ACT AGG GCA GCA

I t

R [Sm

Leu _u Gly ₁₂ Gly ₁ 3Gly ₁₄ Ser ₁ 5Pro Gly Ser Arg Arg and thus the lacZ fusion product of the pTA012 regulatory region is still out of phase with respect to the lacZ reading frame. In order to introduce the N-terminal coding information for the alcohol oxidase structural gene into an open reading frame with the lacZ structural gene, pTA012 was treated with EcoRI-SmaI and the resulting DNA fragments ligated to pSEY101, which had also been previously treated. EcoRI and SmaI to generate the hybrid vector pTA013 (see Figure 30 and the following nucleotide sequence):

G sequence

Rj Sm B) 1.12 kbp | r

GAATTCCC ... CTA GGT GGT GGA TCA CCC GGG GAT CCC GTC GTT CITAAGGG ... GAT CCC CCC CCT AGT GGG CCC CTA GGG CAG CAA t t f

Rj Sm B

LeuuGly ₁₂ Glyj3Gly ₁₄ Ser ₁₅ Pro Gly Asp Pro Val ₉ Val ₁₀ ..β-Galactosidase

The vector pTA013 is then used to transform S. cerevisiae SEY 2102 as described in Figure XV. for further studies described below.

The vector pTA013 is then cleaved with PstI or PstI-NruI and the regulatory region lacZ fusion product contained in the cleaved fragment is ligated into pTAFH1 (see Figure 28), pYJ30 (see Figure 27), or pYA2 (see Figure 23). Fig. 1A) with the HIS4 gene-containing fragment of "commuting" vectors, yielding plasmids pSAOH1, pSAOH5 and pSAOHIO.

pTA013 plus Plasmids have been created pTAFHl pSAOHl pYJ30 pSAOH5 pYA2 pSAOHIO B) Creation of a p76 regulatory area The regulatory region for the lacZ gene fusion product is shown in p76

with the control area, we create it as follows:

1. Applying the entire 5 'region of pPG 6.0.

The 1.35 kb EcoRI fragment of pPG 6.0 was cloned into pSEY101, an E. coli-S. cerevisiae "shuttle" vector, in place of the unique EcoRI, resulting in plasmid pT76U1 (see Figure 30a). The pT6U1 vector is then cleaved with PstI-NruI and the larger DNA fragment ligated with the HIS4 gene-containing fragment of the pTAFH1 "shuttle" vector (Figure 28), as described above for pT76H3 (see Figure 34). Fig. 1A) was ligated with an EcoRl-terminated fragment of the "commuter" vector in the PstI-EcoRI fragment to give pT76H4.

2. 5'-pPG 6.0 Bal31 using a digested product.

The 1.35 kb EcoRI fragment of pPG 6.0 was cloned into pSEY8, an E. coli-S. cerevisiae "shuttle" vector, to the unique EcoRI site, which vector also has a unique SalI site adjacent to the EcoRI site into which Pichia DNA was inserted to give plasmid pTA101 (see Figure 31). Plasmid pTAO1 was cleaved with SalI and treated with Bal31 exonuclease to generate regulatory fragment fragments of p76 polypeptide of various lengths. The blunt-ended fragments are freed from the remainder of the plasmid pSEY8 by digestion with EeoRI. The resulting DNA fragments were cloned into the EcoRI-SmaI linker of pSEY101, yielding, inter alia, plasmid pTAF.85. Plasmid pTAF.85 was selected on the basis of the lacZ gene and the ampicillin resistance gene. (Selection by the lacZ gene is described in Davis, Dibnes, Battey, Basic Methods in Molecular Biology, Elsevier New York, 1986).

The plasmid analog of pTAF.85 is shown in Figure 29

However, it contains a p76 regulatory region instead of a p72 (alcohol oxidase) regulatory region.

The pTAF.85 vector was then treated in an analogous manner to the pTA013 vector to obtain plasmids pTAFH.85, pT76H1 and pT76H2. Thus, the following vector is cleaved and ligated:

pTAF.85 plus: Created plasmids pTAFH1 pTAFH.85 pYJ30 pT76Hl pYA2 pT76H2

XV. example

Regulation of $ galactosidase production in Pichia pastoris

Β-galactosidase production is studied in Pichia pastoris GS115 (NRRL Y-15851) transformants grown in a variety of carbon sources and under different conditions. Transformed strains were grown on minimal medium containing 0.5 pg / ml biotin and 0.1% glucose at 30 ° C until the culture reached the stationary phase. The cells are then harvested by centrifugation and transferred to a minimal medium containing 0.5 pg / ml biotin and 0.5% methanol and grown for 3-5 generations at 30 ° C. Following this initial growth in methanol, the cells are harvested by centrifugation and transferred to fresh minimal medium containing 0.5 pg / ml biotin and either 0.1% glucose or 0.3% methanol as carbon source. Cells are then incubated at 30 ° C for 80 hours, periodically sampled to determine alcohol oxidase and β-galactosidase levels. After about 20-50 hours, the carbon source is depleted and then the cells remain in this carbon source-starved state. Its results are shown in Table I: ·

TABLE 1

Maximum levels of β-galactosidase and alcohol oxidase (unit / ODgoo) (incubation time, hours) in Pichia pastoris NRRL Y-15 851 transformants

PART I

β-galactosidase ³ plasmid 1% glucose glükőzéhezés 0.3% methanol pSAOHl 0 660 (20) 1361 (0) pSAOH5 0.1-0.2 567 (20) 1168 (0) pSAOHIO 0 886 (20) 1559 (0) pTAFH.85 0 0.17 (80) 0.5 pT76Hl 0 3.20 (80) 0 pT76H2 0 not not pT76H3 0 20 781 pT76H4 0.3-0.6 40 (80) 3100

The cells were harvested at different times and assayed for β-galactosidase and alcohol oxidase activity as described in the text.

no - numbers in parentheses indicate incubation time in hours. 60

II. SECTION

Alcohol oxidase ³ plasmid 1% glucose glükózéhezés 0.3% methanol pSAOHl 0 35 530 pSAOH5 0 60 550 pSAOHIO 0 167 425 pTAFH.85 0 not not pT76Hl 0 not not pT76H2 0 not not pT76H3 0 not not pT76H4 0 not not

^a The cell was harvested at different times and β-galactosidase and alcohol oxidase activity was performed as described in the text.

Alcohol oxidase is determined by the dye peroxidase method as described above (see Example VH) and β-galactosidase is determined as follows:

$ -Galaktozidázaktivitás measurement

Final concentration: 0.06 mol / liter 0.04 mol / liter 0.01 mol / liter 0.001 mol / liter 0.05 mol / liter

A) Required solutions:

Z buffer:

Na ₂ HPO ₄ .7H ₂ O 16.1 g '

NaH ₂ PO ₄ 5.5 g KC10.75 g MgSO ₄ .7H ₂ O 0.246 g 2-mercaptoethanol 2.7 ml was charged to 1 liter, adjusted to pH 7. O-Rytrophenyl-QD-Galactoside (ONPG):

400 mg of ONPG (Sigma N-1127) was dissolved in 100 ml of distilled water to make a 4 mg / ml ONPG solution.

B) Determination procedure

1. An aliquot of the culture medium (yeast cell cultures with an OD 0 0-0.5 OD) is centrifuged and the cell pellet is washed with water.

2. Add 1 ml of buffer Z to the cell pellet and mix with 30 µl CHCl ₃ and 30 µl 0.1% SDS, vortex, and incubate for 5 min at 30 ° C.

3. Start the reaction by adding 0.2 mL of ONPG (4 mg / mL) in a Vortex.

4. Stop the reaction by adding 0.1 mL of 1 M Na ₂ CO ₃ at the appropriate time (A _{42 O} <1).

5. Read the absorbance of the supernatant at 420 nm.

C) Calculation of units of β-galactosidase activity: 1 unit = 1 nmole orthonitrophenol (ONP) generated at 30 ° C and pH 7 per minute. 1 nm ONP at 420 nm (A ₄₂₀ ) has an absorbance of 0.0045 at 1 cm path length; Thus, absorbance 1 at 420 nm is 222 nm ONP / ml or 378 nm / 1.7 ml, with a total volume of supernatant to be analyzed of 1.7 ml. The units expressed in the tables are thus calculated as follows:

A420

U = -X378h (min)

HU 205 627 Β

The results in Table I indicate that a foreign protein in a yeast, e.g. β-galactosidase can be produced in Pichia pastoris, either controlled by the presence of methanol in the medium or by carbon starvation after growth on a catabolite-repressing carbon source.

XVI. EXAMPLE

Regulation of $ galactosidase production in S. cervisiae.

Saccharomyces cerevisiae SEY2102, a strain requiring the addition of uracil, leucine and histidine, is transformed with plasmids pTA013 and pT76U1 to survive. Transformed organisms can be readily isolated by selecting colonies that do not require uracil supplementation for growth. Isolated transformants were labeled with SEY2102-pTA013 and SEY2102-pT76Ul. SEY2102-pTA013 was deposited with the Northern Region at the Research Center Institute (Peoris, Illinois) on August 31, 1984 under NRRL Y-15,858.

Cells of NRRL Y-15,858 and SEY2102-pT76U1 were incubated at 30 ° C for 3-4 generations in minimal medium containing 20-20 µg / ml histidine and leucine plus 5% glucose. The cells were then transferred, i.e. harvested by centrifugation, and transferred to YP medium supplemented with 3% carbon source in Table H and grown at 30 ° C for 5 generations. The cells are then incubated for another 50 hours under carbon source starvation conditions and periodically sampled for measurement of β-galactosidase activation. The results are shown in Table II. Table summarizes.

II. SPREADSHEET

β-galactosidase production by S. cerevisiae β-galactosidase (mono- quality / Odeon) A) Alcohol Oxidase Regulatory Area (pTA013) carbon sources 5 generations Starvation conditions after 6 hours 20 hour 50 hours glucose 0.2 105 66 not fructose 0.3 30 31 28 ethanol 23 137 115 77 glycerol 640 806 656 788 galactose 982 960 651 766 B) p76 Regulatory Area (pT76Ul) carbon sources 5 generations Starvation conditions after 12 o'clock 25 hours 30 hours glucose 2.3 254 815 803 glycerol 470 not not not

n = not detected

These results indicate that a yeast foreign protein, i.e., β-galactosidase, can be produced in Saccharomyces cerevisiae under the control of the p72 (alcohol oxidase) and p76 regulatory sites under carbon source conditions and when using a catabolite-repressing carbon source or growth, By growth of S. cerevisiae cells on relatively non-catabolite-repressing carbon sources, e.g. glycerol or galactose.

The level of β-galactosidase expression in S. cerevisiae under the control of the regulatory regions of the present invention can be compared to the expression levels that are possible with other S. cerevisiae regulated promoters.

promotor carbon sources β-galactosidase unit (ODeoo) cytochrome C-lacZ fusion product (CYC1) Raffinose (3%) 460 galactose permease lacZ fusion product (GAL2) Galactose (2%) 450 Invertase-lacZ Fusion Product (SUC2) Glucose (0.1%) 160

It will be seen that the regulatory region of the invention is at least as effective or more effective as a S. cerevisiae promoter than the "native" promoter in S. cerevisiae.

XVII. EXAMPLE

Southern hybridization with yeast genomic DNA

Nine different methanol assimilating strains and one methanol non-assimilating strain were grown on minimal medium (IMI, see Example 1) containing 0.75% methanol and 1% glucose, respectively. Total chromosomal DNA was isolated as described in Figure VI. Thus, the whole nucleic acid was isolated, treated with RNase A, extracted first with phenol-chloroform / isoamyl alcohol, then with chloroform / isoamyl alcohol and finally ethanol precipitated. The precipitated DNA was resuspended in minimal volume of TE buffer and centrifuged to clarify the DNA solution.

Southem hybridization filters are prepared as described in

VI. Thus, 10 µg of total DNA from various yeast species were digested with excess HindIII, electrophoresed, the DNA denatured, the gel neutralized, and the DNA transferred to nitrocellulose filters. The pre-hybridization conditions for these filters are as follows: treated overnight with a solution of 50% deionized formamide, 6 x SSC, 5 x Denhardt's solution, 1 mM EDTA, 0.1% SDS and 100 pg / ml of denatured salmon sperm DNA. The same conditions were applied to the hybridization medium using ³² P-labeled nick translation assays at a final concentration of 10 ⁶ bpm / ml. The test samples include

These include cDNA inserts (PstI fragments) from clones pPC 8.3, pPC 15.0 and pPC 6.7, as well as the 2.7 kbp BglII DNA fragment of the P. pastoris HIS4 gene. Each of these assay samples is applied separately to the same filters for hybridization, which lasts 24 hours at 42 ° C. After hybridization, the filters were washed twice for 15 minutes at room temperature and three times at 65 ° C for 15 minutes in a solution of 2 X SSC, 1 mM EDTA, 0.1% SDS and 0.1 Contains% sodium pyrophosphate. We also try more washes at a lower intensity, for example. At 55 ° C and 42 ° C to confirm the hybridization results. The results of these hybridizations are summarized in Table ÜL.

III. SPREADSHEET

Hybridization of P. pastoris genes to various yeast chromosomal DNA ^x

P. pastoris HIS4 pPC 8.3 pPC 15.0 pPC 6.7 1. P. pastoris NRRL Y-ll 430 + + + + 2. P. pastoris NRRL Y-l 603 + + + + 3. Hansenula capsulatum + + -f- -i- 4.H.henricii + 4- (+) + 5. H. nonfermentans 4- 4- 4 · + 6. H. polymorpha w 4- (+) 4- 7. H. wickerhamii + + + 4- 8. Torulopsis molischiana + + 4- 9. Saccharomyces cerevisiae (+) - - - 10. P. pastoris NRRL Y-15 851 + + + +

* Note: + Hybridization (+) Weak Hybridization

- Hybridization can be observed on the net under the conditions used

The ΠΙ. The results in Table 1A show that the genes of the polypeptides analogous to p76, p72 and p40 are present in virtually all methanol assimilating strains. It is noteworthy that none of these three genes are observed when hybridizing with the methanol non-assimilating strain of S. cerevisiae DNA, whereas homology between the P. pastoris HIS4 gene and the S. cerevisiae HIS4 gene is easily observed.

Bibliography

Nipples and Doly (1979): Nucl. Acids Slit. 7, 15311523.

Douglas M. G. et al. (1984) Proc. Nat. Acad. Sci. USA 81,3983-3987.

Hinnen et al. (1978). Proc. Nat. Acad. Sci. USA 75,1928-1933.

Z. A. Janowicz et al. (1985) Nucl. Acids. Gap. 13.3043-3062.

A. M. Ledeboerés (1985) Nucl. Acids. Gap. 13.3063-3082.

Maxam and Gilbert (1980) Methods in Enzymology

65, 499-560.

Southern (1975) J. Mol. Biol. 98.503-517.

Sanger et al. (1980) J. Mol. Biol. 143

Claims (16)

PATENT CLAIMS A method for producing polypeptides in yeast comprising (a) transforming a yeast host with an expression vector comprising: - a 5'-regulatory region from Pichia pastoris that is sensitive to methanol in the medium and regulates transcription to mRNA, - a polypeptide coding region, - optionally an additional 3 'regulatory region that controls polyadenylation and stopping transcription to mRNA, optionally further one or more bacterial plasmid DNA, bacteriophage DNA, yeast plasmid DNA, yeast chromosomal DNA sequence, and optionally, a marker gene sequence, (b) culturing the transformed host and (c) recovering the expressed polypeptide. 2. The method of claim 1, wherein the host is a yeast strain that can grow on methanol as a carbon and energy source. 3. The method of claim 1 or 2, wherein the host is a yeast strain of the genus Pichia. 4. The method according to any one of claims 1 to 3, wherein the host is Pichia pastoris GS115 (NRRL Y-15851). The method of claim 1, wherein the 5 'regulatory region is a regulatory region derived from Pichia pastoris NRRL Y11430. 6. The method of claim 1, wherein the polypeptide coding region is an alcohol oxidase coding region. 7. The method of claim 1, wherein the polypeptide coding region is a DAS polypeptide production coding region. 8. The method of claim 1, wherein the polypeptide coding region is a region encoding the production of a heterologous polypeptide. 9. The method of claim 8, wherein the polypeptide coding region is a coding region for β-galactosidase production. 10. The method of claim 1, wherein the 5 'regulatory region is transformed with a vector comprising a regulatory region for mRNA transcription encoding alcohol oxidase production. 11. The method of claim 1, wherein the 5 'regulatory region is a DAS polypeptide HU 205 627 Β was transformed with a vector containing a regulatory region for transcription of mRNA. 12. The method of claim 1, wherein the 5 'regulatory region is transformed with a vector comprising a mRNA transcription regulatory region encoding p40 polypeptide production. 13. The method of claim 1, wherein the 5 'regulatory region is transformed with a vector comprising a regulatory region having the following sequence: 5 '-ATGCTTCCAA GATTCTGGTG GGAATACTGC TGATAGCCTA ACGTTCATGA TCAAAATTTA ACTGTTCTAA CCCCTACTTG GACAGGCAAT ATATAAACAG AAGGAAGCTG CCCTGTCTTA AACCTTTTTT TTTATCATCA TTATTAGCTT ACTTTCATAA TTGCGACTGG TTCCAATTGA CAAGCTTTTG ATTTTAACGA CTTTTAACGA CAACTTGAGA AGATCAAAAA ACAACTAATT ATTCGAAACG-3 '. 14. The method of claim 1, wherein the 5 'regulatory region is transformed with a vector comprising a regulatory region having the following sequence: 5 '-AATGGCCCAAA CTGACAGTTT AAACGCTGTC TTGGAACCTA ATATGACAAA AGCGTGATCT CATCCAAGAT GAACTAAGTT TGGTTCGTTG AAATCCTAAC GGCCAGTTGG TCAAAAAGAA ACTTCCAAAA GTCGCCATAC CGTTTGTCTT GTTTGGTATT GATTGACGAA TGCTCAAAAA TAATCTCATT AATGCTTAGC GCAGTCTCTC TATCGCTTCT GAACCCGGTG GCACCTGTGC CGAAACGCAA ATCGGGAAAC AACCCGCTTT TTGGATGATT ATGCATTCTC CTCCACATTGT ATGCTTCCAA GATTCTGGTG GGAATACTGC TGATAGCCTA ACGTTCATGA TCAAAATTTA ACTGTTCTAA CCCCTACTTG GACAGGCAAT ATATAAACAG AAGGAAGCTG CCCTGTCTTA AACCTTTTTT TTTATCATCA TTATTAGCTT ACTTTCATAA TTGCGACTGG TTCCAATTGA CAAGCTTTTG ATTTTAACGA CTTTTAACGA CAACTTGAGA AGATCAAAAA ACAACTAATT ATTCGAAACG-3 '. The method of claim 1, wherein the 5 'regulatory region is transformed with a vector comprising a regulatory region having the following sequence: 5'-BRANCH OF CATCCAAAGA CGAAAGGTTG AATCAAACCT TTTTGCCATC CGACATCCAC AGGTCCATTC TCACACATAA GTGCCAAACG CAACAGGAGG GGATACACTA GCAGCAGACG TTGCAAACGC AGGACTCATC CTCTTCTCTA ACACCATTTT GCATCAAAAC AGCCAGTTAT GGGCTTCATG GAGCTCGCTC ATTCCAATTC CTTCTATTAG GCTACTAACA CCATCACTTT ATTAGCCTGT CTATCCTGGC CCCCCTGGCG AGGTCATG.TT TCTTTATTTC CGAATGCAAC AAGCTCCGCA TTACACCCGA ACATCACTCC AGATCAGGGC TTTCTCAGTG TGGGGTCAAA TAGTTTCATG TTCCCAAATG GCCCAAACT GACAGTTTAA ACGCTCTCTT GGAACCTAAT ATCACAAAAG CGTGATCTCA TCCAAGATGA ACTAAGTTTG GTTCGTTCAA ATCCTAACGG CCAGTTGGTC AAAAAGAAAC TTCCAAAAGT CGCCATACCG TTTGTCTTCT TTGGTATTGA TTGACGAATG CTCAAAAATA ATCTCATTAA TGCTTAGCGC AGTCTCTCTA TCGCTTCTGA ACCCGGTGGC ACCTGTGCCG AAACGCAAAT GGGGAAACAA CCCGCTTTTT GGATGATTAT GCATTCTCCT CCACATTGTA TGCTTCCAAG ATTCTGGTCG GAATACTCCT GATAGCCTAA CGTTCATGAT CAAAATTTAA CTGTTCTAAC CCCTACTTGG ACAGGCAATA TATAAACAGA AGGAAGCTGC CCTGTCTTAA ACCTTTTTTT TTATCATCAT TATTAGCTTA CTTTCATAAT TCCGACTGGT TCCAATTGAC AAGCTTTTGA TTTTAACGAC TTTTAACGAC AACTTGAGAA GATCAAAAAA CAACTAATTA TTCGAAACG-3 '. 16. The method of claim 1, wherein the 5 'regulatory region is transformed with a vector comprising a regulatory region having the following sequence: 5'-TT CACCCATACA ACTATAAACC TTAGCAATTC AAATAACCCC AATTCATTGT TCCGAGTTTA ATATACTTGC CCCTATAAGA AACCAAGGGA TTTCAGCTTC CTTACCCCAT GAACAGAATC TTCCATTTAC CCCCCACTGG AGAGATCCGC CCAAACGAAC AGATAATAGA AAAAAACAAT TCGGACAAAT AGAACACTTT CTCAGCCAAT TAAAGTCATT CCATCCACTC CCTTTAGCTG CCGTTCCATC CCTTTCTTGA GCAACACCAT CGTTAGCCAG TACGAAAGAG GAAACTTAAC CGATACCTTG GAGAAATCTA AGGCGCGAAT GAGTTTAGCC TAGATATCCT TAGTCAAGGG TGTCCGATAC TTCTCCACAT TCAGTCATAG ATGGGCAGCT TCTATCATGA AGAGACGGAA ACGGGCATAA GGGTAACCGC CAAATTATAT AAAGACAACA TGCCCCAGTT TAAAGTTTTT CTTTCCTATT CTTGTATCCT GAGTCACCGT TGTCTTTAAT ATAAAAAGTT CGTTTTAACT TAAGACCAAA ACCAGTTACA ACAAATTATA ACCCCTCTAA ACACTAAAGT TCACTCTTAT CAAACTATCA AACATCAAAA- 3 '